CN103370319B - Novel coelenterazine substrate and using method thereof - Google Patents

Novel coelenterazine substrate and using method thereof Download PDF

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CN103370319B
CN103370319B CN201180062485.1A CN201180062485A CN103370319B CN 103370319 B CN103370319 B CN 103370319B CN 201180062485 A CN201180062485 A CN 201180062485A CN 103370319 B CN103370319 B CN 103370319B
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coelenterazine
pbi
variant
luciferase
ogluc
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CN103370319A (en
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D.H.克劳伯特
P.梅森黑默
J.安奇
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Promega Corp
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Abstract

The polynucleotide of the separation of a kind of luciferase polypeptide encoding modification and novel substrate based on coelenterazine.OgLuc variant polypeptide and SEQ ID NO:1 have at least 60% amino acid sequence identity and have at least one amino acid replacement corresponding to the amino acid whose position in SEQ ID NO:1.The corresponding polypeptide that OgLuc variant polypeptide belongs to luciferase relative to wild type thorn shrimp has at least one in the protein stability of the luminescence of enhancing, the signal stabilization of enhancing and enhancing.

Description

Novel coelenterazine substrate and using method thereof
Cross-Reference to Related Applications
This application claims in the priority of U.S. Provisional Application No. 61/409,422 that on November 2nd, 2010 submits to, its with It is incorporated herein by reference.
Background
Show that the luciferase secreted from deep-sea shrimp thin Fructus Gleditsia acupuncture shrimp (Oplophorus gracilirostris) has perhaps The most interesting feature, and such as high activity, high quantum yield and wide in range substrate specificity (include, such as, coelenterazine (coelenterazine) and multiple coelenterazine analog).When coelenterazine (substrate) and the oxidation of molecular oxygen are belonged to glimmering by stinging shrimp During light element enzyme catalysis, the bioluminescence reaction occurring thorn shrimp to belong to, cause light and the product CO of maximum intensity at 462nm2With Coelenteramide (Shimomura etc., Biochemistry, 17:994 (1978)).Optimal luminescent at 40 DEG C at 0.05- Occur when pH9 in the presence of 0.1M NaCl, and, due to this enzyme unique patience to heat, when using highly purified enzyme, Visible luminescent occurs at a temperature of higher than 50 DEG C, maybe when using partially purified enzyme, occurs when higher than 70 DEG C.? During pH8.7, Shimomura etc. (1978) report natural fluoresence element enzyme has the molecular weight of about 130kDa, it is clear that each include 4 The monomer of 31kDa；When relatively low pH, natural fluoresence element enzyme tends to polymerization.
Recent research has shown that thin Fructus Gleditsia acupuncture shrimp luciferase is the complex of 35kDa and 19kDa native protein, i.e. by Two 19kDa components and the different tetramer of two 35kDa component compositions.Inouye etc. (2000) report coding thorn shrimp and belong to fluorescence The molecular cloning of the cDNA of 35kDa and the 19kDa protein of element enzyme, and the qualification of the protein component of catalytic luminescence reaction.Compile The cDNA of the described protein of code expresses in bacterial cell and mammalian cell, and 19kDa protein is accredited as urging Change the component (Inouye etc., 2000) of the luminous oxidation reaction of coelenterazine.
Thorn shrimp belongs to the 19kDa albumen (GenBank accession number BAB13776,196 aminoacid) of luciferase seemingly to be had There is a minimum catalyst component of luciferase function, and its primary structure and any institute including Imidazopyrazines ketone luciferase The luciferase of report does not have notable similarity (Lorenz etc., PNAS USA, 88:4438 (1991)；Thompson etc., PNASUSA, 86:6567 (1989)).The expression in escherichia coli (E.coli) of the 19kDa protein result in the shape of inclusion body Become (Inouye and Sasaki, Protein Expression andPurification, 56:261-268 (2007)).Inclusion body Formation be likely to be due to the unstability of protein and cause.
Thorn shrimp belong to the substrate specificity of luciferase unexpectedly extensively (Inouye and Shimomura.BBRC223: 349(1997)).Such as, double deoxidation coelenterazine (bisdeoxycoelenterazine) (that is, coelenterazine-hh), a kind of coelenterazine Analog, be suitable with coelenterazine thorn shrimp belong to luciferase splendid substrate (Nakamura etc., Tetrahedron, Lett., 38:6405 (1997)).Further, thorn shrimp belongs to the enzyme that luciferase is secretion, with marine facies mussel-shrimp cypridina hilgendorfii (Cypridina (Vargula) hilgendorfii) luciferase similar (Johnson and Shimomura, Meth.Enzyme, 57:331 (1978)), it also uses Imidazopyrazines ketone type fluorescence the most luminous.
General introduction
On the one hand, it relates to formula (Ia) or the compound of (Ib):
Or
Wherein R2Group selected from consisting of:
Or C2-5Directly Alkyl group；
R6Choosing freely-H ,-OH ,-NH2,-OC (O) R or-OCH2The group of OC (O) R composition；
R8Group selected from consisting of:
H or low-grade cycloalkyl；
Wherein R3And R4It is H or is C1-2Alkyl；
W is-NH2, halo ,-OH ,-NHC (O) R ,-CO2R；
X is-S-,-O-or-NR22-；
Y is-H ,-OH or-OR11；
Z is-CH-or-N-；
Each R11Independently be-C (O) R " or-CH2OC(O)R”；
R22For H, CH3Or CH2CH3；
Each R independently be C1-7Straight chained alkyl or C1-7Branched alkyl；
R " it is C1-7Straight chained alkyl or C1-7Branched alkyl；
The key table of dotted line shows the existence of optional ring, and it can be saturated or unsaturated；
Precondition is to work as R2ForOr Time, R8It is not
Precondition is to work as R2ForTime, R8It isOr low-grade cycloalkyl；With
Precondition is to work as R6For NH2Time, R2For,Or C2-5Alkyl；
Or R8It is not
On the other hand, disclosure relates to selected from following compound:
On the one hand, it relates to the compound of below formula:
Or
On the one hand, it relates to coding and SEQ ID NO:1 have at least 60% amino acid sequence identity The polynucleotide of the separation of OgLuc variant polypeptide, described OgLuc variant polypeptide is corresponding to the aminoacid in SEQ ID NO:1 Position include that at least one amino acid replacement, wherein said OgLuc variant polypeptide have the luminescence of enhancing.
On the one hand, it relates to coding and SEQ ID NO:1 have at least 60% amino acid sequence identity The polynucleotide of the separation of OgLuc variant polypeptide, described OgLuc variant polypeptide is corresponding to the aminoacid in SEQ ID NO:1 Position include at least one amino acid replacement, wherein said OgLuc variant polypeptide is relative to the OgLuc of SEQ ID NO:3 Polypeptide has the luminescence of enhancing, precondition be by the polypeptide of polynucleotide encoding be not in table 47 listed those wherein it One.
On the one hand, it relates to coding and SEQ ID NO:1 have at least 60% amino acid sequence identity The polynucleotide of the separation of OgLuc variant polypeptide, described OgLuc variant polypeptide is corresponding to the aminoacid in SEQ ID NO:1 Position include at least one amino acid replacement, the wherein said OgLuc variant polypeptide polypeptide relative to SEQ ID NO:31 Having the luminescence of enhancing, precondition is not for SEQ ID NO:3 or 15 by the polypeptide of polynucleotide encoding.
On the one hand, it relates to coding and SEQ ID NO:1 have at least 60% amino acid sequence identity The polynucleotide of the separation of OgLuc variant polypeptide, described OgLuc variant polypeptide is corresponding to the aminoacid in SEQ ID NO:1 Position include at least one amino acid replacement, the wherein said OgLuc variant polypeptide polypeptide relative to SEQ ID NO:29 Having the luminescence of enhancing, precondition is not for SEQ ID NO:3 or 15 by the polypeptide of polynucleotide encoding.
On the one hand, it relates to the OgLuc polypeptide of coding and SEQ ID NO:1 has at least 80% aminoacid sequence The polynucleotide of the separation of the OgLuc variant polypeptide of homogeneity, described OgLuc variant polypeptide includes corresponding to SEQ ID NO:1 Amino acid replacement A4E, Q11R, A33K, V44I, P115E, Q124K, Y138I, N166R, I90V, F54I, Q18L, F68Y, L72Q and M75K, and described OgLuc variant polypeptide has uciferase activity.
On the one hand, it relates to the OgLuc polypeptide of coding and SEQ ID NO:1 has at least 80% aminoacid sequence The polynucleotide of the separation of the OgLuc variant polypeptide of homogeneity, the aminoacid of wherein correspond to SEQ ID NO:1 the 4th is Glutamic acid, the aminoacid of the 11st is arginine, and the aminoacid of the 18th is leucine, and the aminoacid of the 33rd is lysine, The aminoacid of the 44th is isoleucine, and the aminoacid of the 54th is isoleucine, and the aminoacid of the 68th is tyrosine, the 72nd The aminoacid of position is glutamine, and the aminoacid of the 75th is lysine, and the aminoacid of the 90th is valine, the 115th Aminoacid is glutamic acid, and the aminoacid of the 124th is lysine, and the aminoacid of the 138th is isoleucine, and the 166th Aminoacid is arginine, and described OgLuc variant polypeptide has uciferase activity.
On the one hand, it relates to the OgLuc polypeptide of coding and SEQ ID NO:1 has at least 80% aminoacid sequence The polynucleotide of the separation of the OgLuc variant polypeptide of homogeneity, described OgLuc variant polypeptide includes corresponding to SEQ ID NO:1 Amino acid replacement A4E, Q11R, A33K, V44I, P115E, Q124K, Y138I, N166R, Q18L, F54I, L92H and Y109F, And described OgLuc variant polypeptide has uciferase activity.
On the one hand, it relates to the OgLuc polypeptide of coding and SEQ ID NO:1 has at least 80% aminoacid sequence The polynucleotide of the separation of the OgLuc variant polypeptide of homogeneity, described OgLuc variant polypeptide includes corresponding to SEQ ID NO:1 Amino acid replacement A4E, Q11R, A33K, V44I, A54I, F77Y, I90V, P115E, Q124K, Y138I and N166R, and OgLuc variant polypeptide has uciferase activity.
On the one hand, it relates to the OgLuc polypeptide of coding and SEQ ID NO:1 has at least 80% aminoacid sequence The polynucleotide of the separation of the OgLuc variant polypeptide of homogeneity, the aminoacid of wherein correspond to SEQ ID NO:1 the 4th is Glutamic acid, the aminoacid of the 11st is arginine, and the aminoacid of the 18th is leucine, and the aminoacid of the 33rd is lysine, The aminoacid of the 44th is isoleucine, and the aminoacid of the 54th is isoleucine, and the aminoacid of the 92nd is histidine, the The aminoacid of 109 is phenylalanine, and the aminoacid of the 115th is glutamic acid, and the aminoacid of the 124th is lysine, the 138th The aminoacid of position is isoleucine, and the aminoacid of the 166th is arginine, and OgLuc variant polypeptide has luciferase and lives Property.
On the one hand, it relates to the OgLuc polypeptide of coding and SEQ ID NO:1 has at least 80% aminoacid sequence The polynucleotide of the separation of the OgLuc variant polypeptide of homogeneity, wherein the 4th amino acids corresponding to SEQ ID NO:1 is paddy Propylhomoserin, the 11st amino acids are arginine, and the 33rd amino acids is lysine, and the 44th amino acids is isoleucine, the 54th Aminoacid is isoleucine, and the 77th amino acids is tyrosine, and the 90th amino acids is valine, and the 115th amino acids is paddy Propylhomoserin, the 124th amino acids is lysine, and the 138th amino acids is isoleucine, and the 166th amino acids is arginine, and OgLuc variant polypeptide has uciferase activity.
On the one hand, it relates to include the many nucleoside encoding the separation of the polynucleotide of the polypeptide of SEQ ID NO:19 Acid.
On the one hand, it relates to include SEQ ID NO:18, SEQ ID NO:24, SEQID NO:25, SEQ ID The polynucleotide of the separation of the polynucleotide of NO:42, SEQ ID NO:88 or SEQ ID NO:92.
On the one hand, it relates to coding and SEQ ID NO:1 have the ten of at least 30% amino acid sequence identity The polynucleotide of the separation of foot class luciferase polypeptide, described polypeptide includes the sequence mould of the sequence pattern corresponding to formula (VII) Formula and include less than 5 differences, wherein difference includes according to the OgLuc pattern listed in table 4 pattern relative to formula (VII) The difference of position 1,2,3,5,8,10,12,14,15,17 or 18, and according to OgLuc pattern listed in table 4 at formula (VII) Any mode position between room or insertion, wherein said decapods luciferase produces luminescence in the presence of coelenterazine.
On the one hand, it relates to encode the nucleotide sequence of the synthesis of OgLuc variant polypeptide, described OgLuc variant Polypeptide include with the parent nucleic acid sequence with SEQ ID NO:2 have 80% or lower nucleic acid sequence identity and and SEQ It is same that ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25 have 90% or higher nucleotide sequence The fragment of at least 100 nucleotide of one property or its complement, the sequence iden of wherein said reduction is the nucleotide of synthesis The result of codons different relative to the codon in parent nucleic acid sequence in sequence, the nucleotide sequence of wherein said synthesis Encode and the OgLuc by the corresponding luciferase coded by parent nucleic acid sequence with at least 85% amino acid sequence identity Variant, and the nucleotide sequence of wherein said synthesis has the regulation and control sequence of number of minimizing relative to described parent nucleic acid sequence Row.
On the one hand, it relates to encode the nucleotide sequence of the synthesis of OgLuc variant polypeptide, described OgLuc variant Polypeptide include with the parent nucleic acid sequence with SEQ ID NO:14 have 80% or lower nucleic acid sequence identity and and SEQ ID NO:22 or SEQ IDNO:23 has the sheet of at least 300 nucleotide of at least 90% or higher nucleic acid sequence identity Section or its complement, the sequence iden of wherein said reduction be described synthesis nucleotide sequence in relative to described parental generation core The result of the codon that codon in acid sequence is different, wherein said synthesis nucleotide sequence coded with by described parental generation core Corresponding luciferase coded by acid sequence has a LUC Photinus pyralis LUC Photinus pyralis FL of at least 85% amino acid sequence identity, and wherein The nucleotide sequence of described synthesis has the regulating and controlling sequence of the number of reduction relative to described parent nucleic acid sequence.
On the one hand, it relates to encode the nucleotide sequence of the synthesis of OgLuc variant polypeptide, described OgLuc variant Polypeptide include with the parent nucleic acid sequence with SEQ ID NO:18 have 80% or lower nucleic acid sequence identity and and SEQ ID NO:24 or SEQ IDNO:25 have at least 100 nucleotide of 90% or higher nucleic acid sequence identity fragment or Its complement, the sequence iden of wherein said reduction be described synthesis nucleotide sequence in relative to described parent nucleic sequence The result of the codon that codon in row is different, wherein said synthesis nucleotide sequence coded with by described parent nucleic sequence Corresponding luciferase coded by row has the OgLuc variant of at least 85% amino acid sequence identity, and wherein said conjunction The nucleotide sequence become has the regulating and controlling sequence of the number of reduction relative to described parent nucleic acid sequence.
On the one hand, it relates to the fusogenic peptide of the signal peptide from thin Fructus Gleditsia acupuncture shrimp with heterologous protein, wherein Described signal peptide is SEQ ID NO:54, and wherein said fusogenic peptide is expressed and from this emiocytosis in cell.
On the one hand, it relates to generate the method for polynucleotide of coding OgLuc variant polypeptide, described method bag Include: (a) utilizes the parental fusion protein constructs including parental generation OgLuc polypeptide and at least one heterologous polypeptide to melt to generate variant The library of hop protein；(b) this library screening is had relative to parental fusion protein constructs the enzyme of the luminescence of enhancing, enhancing At least one in the biocompatibility of stability or enhancing.
On the one hand, it relates to generate the coding codon optimized polynucleotide for the luciferase of organism Method, described method includes: for each aminoacid in luciferase, and the most frequently used two kind used from organism are close Numeral randomly chooses codon to encode described aminoacid to produce the polynucleotide that first password optimizes.
Other aspects of the present invention will be by considering that the detailed description and the accompanying drawings become obvious.
Accompanying drawing is sketched
Fig. 1 shows natural coelenterazine, known double-coelenterazine (coelenterazine-hh) and the chemistry of known coelenterazine-h Structure, wherein R2, R6 and R8 represent the region of the molecule making modification.
Fig. 2 shows novel coelenterazine PBI-3939, PBI-3889, PBI-3945, PBI-4002, PBI-3841, PBI- 3897, the chemical constitution of PBI-3896, PBI-3925, PBI-3894, PBI-3932 and PBI-3840.
Fig. 3 shows that the Km of PBI-3939 determines.
Fig. 4 shows the chemical constitution of the multiple novel coelenterazine of the present invention.
Fig. 5 A-G shows and utilizes natural, known and novel coelenterazine as substrate from the expression C1+A4E of cracking Bacterial cell in generate luminescence (RLU).Fig. 5 A, 5C-5G show and utilize natural coelenterazine as a comparison, about known The luminous independent experiment in units of RLU generated by C1+4AE is measured with novel coelenterazine.Fig. 5 B show with Natural coelenterazine compares, and utilizes the luminous decline at double that in Fig. 5 A, the substrate of display is generated by C1+4AE.
Fig. 6 A-D shows and utilizes natural coelenterazine (" Coelente "), known coelenterazine-h (" h "), it is known that coelenteron Element-hh (" h, h "), it is known that 2-methyl coelenterazine (" 2-me "), it is known that coelenterazine-v (" v ") and novel coelenterazine PBI- 3840、PBI-3897、PBI-3889、PBI-3899、PBI-3900、PBI-3912、PBI-3913、PBI-3925、PBI-3897、 PBI-3899、PBI-3889、PBI-3939、PBI-3933、PBI-3932、PBI-3946、PBI-3897、PBI-3841、PBI- 3896, PBI-3925 with PBI-3945 generates from the bacterial cell expressing different OgLuc variants of cracking as substrate Luminous.
Fig. 7 shows the amino acid replacement in different OgLuc variants.
Fig. 8 A-B shows and utilizes natural coelenterazine (" Coelenterazine "), known coelenterazine-h (" H "), known Coelenterazine-hh (" h, h ") and novel coelenterazine PBI-3840, PBI-3925, PBI-3912, PBI-3889, PBI-3939, PBI-3933, PBI-3932, PBI-3946, PBI-3941 and PBI-3896 as substrate from cracking expressions Fig. 7 listed by The luminescence generated in the bacterial cell of OgLuc variant.
Fig. 9 shows and utilizes natural coelenterazine (" coelenterazine "), known coelenterazine-hh (" h, h ") and novel coelenterazine PBI-3939, PBI-3945, PBI-3840, PBI-3932, PBI-3925, PBI-9894 and PBI-3896 as substrate from cracking The bacterial cell expressing different OgLuc variant in the luminescence that generates.
Figure 10 shows the amino acid replacement in different OgLuc variants.
Figure 11 shows and utilizes natural coelenterazine (" coelenterazine "), known coelenterazine-hh (" h, h ") and novel coelenterazine PBI-3939, PBI-3945, PBI-3840, PBI-3932, PBI-3925, PBI-3894 and PBI-3896 as substrate from cracking Expression Figure 10 in listed OgLuc variant bacterial cell in the luminescence that generates.
Figure 12 shows and utilizes natural coelenterazine (" coelenterazine "), known coelenterazine-hh (" h, h ") and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, PBI-3840, PBI-3932, PBI-3925, PBI-3894, PBI-3896 and PBI- 3897 luminescences generated from the bacterial cell of the expression OgLuc variant of cracking as substrate.
Figure 13 shows and utilizes natural coelenterazine (" coelenterazine "), known coelenterazine-hh (" H, H ") and novel coelenterazine PBI-3897, PBI-3896 and PBI-3894 are as sending out that substrate generates from the bacterial cell of the expression OgLuc variant of cracking Light.
Figure 14 shows the amino acid replacement in different OgLuc variants.
Figure 15 shows and utilizes natural coelenterazine (" coelenterazine "), known coelenterazine-hh (" h, h ") and novel coelenterazine PBI-3897, PBI-3841, PBI-3896 and PBI-3894 as substrate from cracking expressions Figure 14 listed by OgLuc variant Bacterial cell in generate luminescence.
Figure 16 show utilize natural coelenterazine (" coelenterazine "), known coelenterazine-h (" H "), known coelenterazine- Hh (" HH ") and novel coelenterazine PBI-3841 and PBI-3897 as substrate from the bacterial cell of the expression OgLuc variant of cracking The luminescence of middle generation.
Figure 17 shows and utilizes natural coelenterazine (" Coel "), known coelenterazine-hh (" h, h ") and novel coelenterazine PBI-3897 and PBI-3841 belongs to (Renilla) fluorescence as substrate from expression OgLuc variant and the humanized sea pansy of cracking The luminescence generated in the bacterial cell of element enzyme (hRL).
Figure 18 shows and utilizes natural coelenterazine (" coelenterazine "), known coelenterazine-hh (" h, h ") and novel coelenterazine PBI-3939, PBI-3945, PBI-3889 and PBI-4002 as substrate from the antibacterial expressing different OgLuc variants of cracking The luminescence generated in cell.
Figure 19 shows and utilizes natural coelenterazine (" coelenterazine "), it is known that coelenterazine-h (" H "), it is known that coelenterazine- Hh (" h, h ") and novel coelenterazine PBI-3939, PBI-3945, PBI-3889 and PBI-4002 as substrate from the expression of cracking The luminescence generated in the bacterial cell of different OgLuc variants.
Figure 20 shows the amino acid replacement in OgLuc variant.
Figure 21 shows and utilizes natural coelenterazine (" coelenterazine "), known coelenterazine-h (" H "), it is known that coelenterazine- Hh (" h, h ") and novel coelenterazine PBI-3939, PBI-3945, PBI-4002, PBI-3932 and PBI-3840 as substrate from The luminescence generated in the bacterial cell of OgLuc variant listed in expression Figure 20 of cracking.
Figure 22 shows the amino acid replacement in different OgLuc variants.
Figure 23 show utilize natural coelenterazine (" coelenterazine "), known coelenterazine-h (" H "), known coelenterazine- Hh (" h, h ") and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, PBI-4002, PBI-3932 and PBI-3840 make For the luminescence of generation in the bacterial cell of the OgLuc variant listed from expressions Figure 22 of cracking of substrate.
Figure 24 show utilize natural coelenterazine (" coelenterazine "), known coelenterazine-h (" H "), known coelenterazine- Hh (" H, H ") and novel coelenterazine PBI-3939 and PBI-3945 as substrate from cracking express different OgLuc variants and The luminescence generated in the bacterial cell of hRL (" sea pansy genus ").
Figure 25 utilizes natural coelenterazine (" coelenterazine "), known coelenterazine-hh (" h, h ") and novel coelenterazine PBI- 3939, PBI-3945, PBI-3889 and PBI-4002 as substrate from the bacterial cell expressing different OgLuc variants of cracking The luminescence of middle generation.
Figure 26 shows the amino acid replacement in different OgLuc variants.
Figure 27 show utilize natural coelenterazine (" coelenterazine "), known coelenterazine-h (" H "), known coelenterazine- Hh (" h, h ") and novel coelenterazine PBI-3939, PBI-3945, PBI-3889 and PBI-4002 as substrate from the expression of cracking The luminescence generated in the bacterial cell of OgLuc variant listed in Figure 26.
Figure 28 shows and utilizes natural coelenterazine (" Coel. "), known coelenterazine-h (" H "), known coelenterazine-hh (" H, H ") and novel coelenterazine PBI-3939, PBI-3945, PBI-3889 and PBI-4002 are as substrate from the expression of cracking not The luminescence generated in same OgLuc variant and the bacterial cell of hRL (" sea pansy genus ").
Figure 29 show utilize natural coelenterazine and PBI-3939 as the 9B8opt in substrate bacterial lysate and The luminescence of 9B8opt+K33N, and compared with natural coelenterazine these variants for the relative specificity of PBI-3939.
Figure 30 A-D shows and utilizes natural coelenterazine (Figure 30 A), coelenterazine-h (Figure 30 B) and PBI-399 (Figure 30 C) to exist Mutation analysis at 166th.
Figure 31 shows the luminescence of the different disappearance in OgLuc variant L27V, wherein (-) it is machine background.
Figure 32 shows and utilizes natural coelenterazine as substrate from the HEK293 cell of the expression hRL (" sea pansy genus ") of cracking The standardized luminescence of middle generation, utilize fluorescein (BRIGHT-GLOTMMeasure reagent) as substrate from cracking expression Luciola vitticollis In the HEK293 cell of luciferase (Luc2) generate standardized luminescence and utilize novel PBI-3939 as substrate from The standardized luminescence generated in the HEK293 cell expressing different OgLuc variants of cracking.
Figure 33 shows the signal utilizing novel coelenterazine PBI-3945 as IV and 15C1 in substrate bacterial lysate Stability and utilize novel coelenterazine PBI-3889 as the signal stabilization of IV and 9B8 in substrate bacterial lysate.
Figure 34 A-B show belong to luciferase (Rluc) with LUC Photinus pyralis LUC Photinus pyralis FL (Fluc) and sea pansy compared with OgLuc The higher activity (Figure 34 A) of variant L27V and signal stabilization (Figure 34 B).
Figure 35 shows and utilizes novel coelenterazine PBI-3939 as OgLuc variants different in substrate bacterial lysate Vmax (RLU/ second) and Km (μM) value.
Figure 36 shows and utilizes novel coelenterazine PBI-3939 as OgLuc variants different in substrate bacterial lysate Vmax (RLU/ second) and Km (μM) value.
Figure 37 shows and utilizes novel coelenterazine PBI-3939 as 9B8opt and 9B8opt+ in substrate bacterial lysate The Vmax (RLU/ second) of both K33N and Km (μM) value.
Figure 38 shows and utilizes natural coelenterazine as the different OgLuc variant in substrate bacterial lysate when 50 DEG C Protein stability, luminescence at t=0 and by minute in units of half-life.
Figure 39 A-B show belong to luciferase (hRL) and LUC Photinus pyralis LUC Photinus pyralis FL (Luc2) with sea pansy compared with at 25 DEG C The structural intergrity of OgLuc variants different in bacterial lysate under (Figure 39 A) and 37 DEG C (Figure 39 B) is (by expression, stability Determine with dissolubility, as shown in analyzed by SDS-PAGE).
Figure 40 A-B show utilize novel coelenterazine PBI-3939 as the 9B8opt in substrate bacterial lysate and 9B8opt+K33N protein stability at 60 DEG C, elapses natural logrithm (the ln) (figure of luminescence (in units of RLU) in time 40A) and by hour in units of half-life (Figure 40 B).
Figure 41 shows the percentage ratio of the activity of OgLuc variant 9B8 and L27V at 60 DEG C.
Figure 42 A-B shows that OgLuc variant L27V's under different pH (Figure 42 A) and salinity (Figure 42 B) is protein stabilized Property.
Figure 43 A-B shows C1+A4E (Figure 43 A) and the analysed by gel filtration chromatography of 9B8 (Figure 43 B) of purification.
Figure 44 shows the analysed by gel filtration chromatography showing that OgLuc variant L27V exists with monomeric form.
Figure 45 A-B shows and analyzes (Figure 45 A) by SDS-PAGE, the undiluted and bacterial lysate sample of 1: 1 dilution Different OgLuc variant in product-(HT7) protein expression level of fusion protein, and standardized albumen Expression (Figure 45 B).
Figure 46 A-B shows protein expression (Figure 46 A) and the dissolubility (figure of OgLuc variant 9B8opt, V2 and L27V 46B)。
Figure 47 show utilize natural coelenterazine and novel coelenterazine PBI-3939 as substrate from cracking expression IV, The standardized luminescence in units of RLU generated in the HEK293 cell of 9B8 and hRL (" sea pansy genus ").
Figure 48 shows and is utilized respectively PBI-3939, Luciferin (BRIGHT-GLOTMMeasure reagent) and natural coelenterazine As substrate from expression pF4Ag-Ogluc-9B8-HT7, pF4Ag-Luc2-HT7 and pF4Ag-Renilla-HT7 of cracking The standardized luminescence in units of RLU generated in HEK293 cell.
Figure 49 show utilize novel coelenterazine PBI-3939 as substrate, from the expression coding 9B8opt of cracking or The luminescence generated in the HEK293 cell of the plasmid DNA of 30ng or 100ng of 9B8opt+K33N (" K33N ").
Figure 50 A-E shows that (Figure 50 B) is glimmering with Lampyridea in HEK293 cell (Figure 50 A) and HeLa cell (fusion) Light element enzyme (Luc2) compares the luminescence of OgLuc variant L27V, in HEK293 (" HEK ") and HeLa cell (" HeLa "), with OgLuc variant L27V (Figure 50 C) compares with LUC Photinus pyralis LUC Photinus pyralis FL (Luc2) (Figure 50 D)Sending out of fusant Light, and with-LUC Photinus pyralis LUC Photinus pyralis FL (Luc2) comparesThe protein expression of L27V.
Figure 51 shows that the inhibition analysis for LOPAC library OgLuc variant 9B8 and L27V is to determine that it is for phase of missing the target The sensitivity of interaction.
Figure 52 A-E shows the inhibition analysis (figure to OgLuc variant 9B8 and L27V by suramin and Tyr ag835 52A-C) and suramin (Figure 52 D) and the chemical constitution of Tyr ag835 (Figure 52 E).
Figure 53 show analysis in the presence of BSA the activity of OgLuc variant 9B8 and L27V to determine nonspecific proteins The resistance interacted.
Figure 54 shows that the percent activity of OgLuc variant 9B8 and L27V is to determine the reactivity to plastics.
Figure 55 shows that the known coelenterazine-h of utilization is using at forskolin compared with hRL (" sea pansy genus ") as substrate From the HEK293 of expression IV cAMP transcribed reporter cracked when reason (" induction ") and unused forskolin process (" substrate ") The luminescence generated in cell, and owing to forskolin processes multiple induction (response) (" multiple ") caused.
Figure 56 shows and utilizes PBI-3939 (for 9B8 and 9B8opt), natural coelenterazine (for hRL) or fluorescein (BRIGHT-GLOTMMeasure reagent；For Luc2) processing ("+FSK ") or the process of unused forskolin as substrate with forskolin Turn from expression 9B8,9B8opt, hRL (" sea pansy genus ") or LUC Photinus pyralis LUC Photinus pyralis FL (" the Luc2 ") cAMP of cracking time ("-FSK ") The standardized luminescence generated in the HEK293 cell of record report, and owing to forskolin processes multiple induction (response) caused (" multiple ").
Figure 57 shows and utilizes novel coelenterazine PBI-3939 processing (" induction ") or not as substrate with forskolin Transcribe from expression 9B8opt and 9B8opt+K33N (" the K33N ") cAMP of cracking in the case of processing (" substrate ") with forskolin The luminescence generated in the HEK293 cell of report, and owing to forskolin processes multiple induction (" multiple induction ") caused.
Figure 58 A-C shows the OgLuc variant 9B8 about the number of ways in various kinds of cell type and L27V cracking performance report Accuse the luminescence of sub-construct.
Figure 59 A-C shows in different cell line and utilizes different response element OgLuc variant L27V report The luminescence of construct.
Figure 60 A-B shows and has CMV promoter (Figure 60 A) or the elongated long ascites of NFkB response element (Figure 60 B) Flea (Metridia longa) luciferase compare OgLuc variant L27V can secrete report son luminescence.
Figure 61 A-F shows compared with the L27V (L27V00) expressed in HeLa cell, the form of the optimization of L27V The absolute luminescence (Figure 61 A and 61B) of (L27V01, L27V02 and L27V03), standardized luminescence (Figure 61 C and 61D) and multiple Response (Figure 61 E and 61F).
Figure 62 A-B shows the secreting type expressed in the HepG2 cell that the rhTNF α (" TNF α ") by various dose processes OgLuc variant L27V02 (containing IL-6 secretion signal) report son (Figure 62 A) and L27V02 (" L27V (02) "), L27V02P The luminescence of (" L27V (02) P (01) ") and luc2 (" Fluc ") report.
Figure 63 shows and utilizes natural coelenterazine as substrate containing secretory signal sequence or without secretory signal sequence HRL (" sea pansy genus ") compares and utilizes novel PBI-3939 as substrate from expressing containing secretory signal sequence or without secretion signal The luminescence generated in the culture medium of the HEK293 cell of the codon optimized variant IVopt of sequence and lysate sample.
Figure 64 A-D shows the secretion expressed in Chinese hamster ovary celI (Figure 64 A and 64B) and HeLa cell (Figure 64 C and 64D) The luminescence of type OgLuc variant 9B8, V2 and L27V report.
Figure 65 A-B show the luminescence that utilizes PBI-3939 to produce from secreting type OgLuc variant 9B8 and V2 as substrate with Utilize Ready-to-GlowTMAs (Figure 65 A) on the luminous digital of the secreting type luciferase of the elongated long ascites flea of substrate and figure The comparison of (Figure 65 B) on table.
Figure 66 A-B shows and utilizes coelenterazine derivant ENDURENTM(Figure 66 A) and VIVIRENTM(Figure 66 B) and novel Coelenterazine PBI-3939 (Figure 66 B) is carried on the back from being higher than of HEK293 Hemapoiesis expressing hRL (" Ren ") and 9B8opt as substrate The luminous multiple of scape increases.
Figure 67 A-D shows transient expressionFusant (Figure 67 A) or IL6-L27V fusant (figure The Confocal Images of U2OS cell 67B-D).Scale=20 μm.
Figure 68 shows and utilizes natural coelenterazine in sandwich background existence (" Sand ") or not exist as substrate (pF4Ag) luminescence generated from the bacterial cell expressing different OgLuc variants and hRL (" sea pansy genus ") of cracking under.
Figure 69 shows and utilizes natural coelenterazine as substrate owing to the existence of sandwich background causes different OgLuc to become The multiple of the activity of body and hRL (" sea pansy genus ") reduces.
Figure 70 shows and utilizes novel coelenterazine PBI-3939 as substrate owing to the existence of sandwich background causes antibacterial In lysate, the multiple of the activity of 9B8opt and 9B8opt+K33N reduces.
Figure 71 shows the spectral profile figure of OgLuc variant L27V.
OgLuc variant L27V when Figure 72 shows at non junction or has 5,10 or 20 amino acid whose joints, The luminescence of (CP) form of two circulation exchanges of CP84 and CP95.
Figure 73 A-G shows wheat germ extract (Figure 73 A-D), escherichia coli (Figure 73 F-G) and HEK293 cell (Figure 73 H) The luminescence of the different CP-TEV protease L27V construct of middle expression.Figure 73 A-D showed before TEV adds different The substrate of CP-TEV protease L27V construct is luminous.Figure 73 E shows the CP-TEV protease L27V construct of Figure 73 A-D Response.
Figure 74 shows the multiple response of different complementary action of protein L27V pair.
Figure 75 A-C shows the luminescence of different complementary actions of protein (PCA) L27V pair, described complementary action of protein L27V pair That is: 1/4 configuration (Figure 75 A) or 2/3 configuration (Figure 75 B) is utilized a L27V fragment of every pair to be merged with FKBP or FRB, and The interaction of FKBP and FRB monitored in HEK293 cell.Also monitor different complementary action of protein (PCA) negative controls Luminescence (Figure 75 C).
Figure 76 A-H shows the luminescence of different complementary actions of protein (PCA) L27V pair, described complementary action of protein L27V pair That is: utilize 2/3 configuration (Figure 76 A and 76C) or 1/4 configuration (Figure 76 B and 76D) by the L27V fragment of every pair and FKBP or FRB merges, and wheat germ extract (Figure 76 A and 76B) and rabbit reticulocyte lysate (RRL) (Figure 76 C and 76D) are middle is monitored The interaction of FKBP and FRB.Sending out of different complementary action of protein (PCA) negative controls is also measured in cell free system Light (Figure 76 E).1/4 configuration is in cell free system (Figure 76 F), HEK293 cell (Figure 76 G) and cracking system (Figure 76 H).
Figure 77 A-C shows that the different complementary action of protein L27V processed with FK506 and rapamycin (Figure 77 A) is to sending out Light, and FK506 (Figure 77 A) and the chemical constitution of rapamycin (Figure 77 B).
Figure 78 shows the activity utilizing forskolin to process OgLuc variant 9B8cAMP biosensor.
Figure 79 A-D shows that in rabbit reticulocyte lysate (Figure 79 A-B) and HEK293 cell (Figure 79 C-D), circulation is handed over (Figure 79 A and 79C) and the luminescence of (Figure 79 B and 79D) L27V variant of amitosis changed.
Figure 80 A-B shows the OgLuc variant L27V (Figure 80 A) in U2OS cell and control vector pGEM3ZF (Figure 80 B) Subcellular proteomics about different open-assembly times.
Figure 81 A-C show with do not merge L27V comparison (Figure 81 A) compared with, with transcription factor Nrf2 (Figure 81 B) or The Subcellular Localization of the OgLuc variant L27V that GPCR (Figure 81 C) merges.
Figure 82 A-C shows and utilizes PBI-4377OgLuc variant 9B8opt for monitoring the purposes of intracellular signaling pathway (Figure 82 A).9B8opt luciferase merges with IkB (Figure 82 B) or ODD (the degrading texture territory of the oxygen dependence of Hif-1 α), and leads to Cross the luminous monitoring multiple response for stimulus object (be TNF α for IkB, and be phenanthroline for ODD).
Figure 83 A-C shows that use OgLuc variant (Figure 83 A), L27V02 (Figure 83 B) or sea pansy belong to luciferase (Rluc) (Figure 83 C) monitors oxidative stress signal pathway.
Figure 84 A-B shows that Nrf2-L27V02 sensor (Figure 84 A) and Nrf2 (ARE)-Luc2P report son (Figure 84 B) Relatively.
Figure 85 A-B show utilize 1 μM of TMR (Figure 85 A) or 10 μMs of rhodamines 110 (Figure 85 B) as the part of HT7 and Utilize coelenterazine-h as the substrate of IV, the emission spectrum of IV-HT7 in the case of there is part and do not have part.
Figure 86 shows and utilizes pre-coelenterazine substrate from mixing ("+caspase ") with caspase-3 mRNA or not with half The luminescence generated in the bacterial cell of the expression 9B8opt of the cracking of Guang aspartase-3 mixing (" without caspase ").
Figure 87 A-C shows and utilizes PBI-3939 as substrate mould by rapamycin treatment (Figure 87 B) or unused thunder handkerchief The luminescence generated from circulation exchange, amitosis L27V variant CP84 and CP103 when element processes (not shown), and due to The response (Figure 87 C) that rapamycin treatment causes.The concept of the variant of the amitosis of circulation exchange is shown in Figure 87 A.
Figure 88 shows the residual activity percentage ratio of L27V variant after the carbamide being exposed to different amounts.
Figure 89 shows the effect to the activity of L27V variant of the 3M carbamide.
Figure 90 A-B shows that the nuclear receptor (NR) of the OgLuc fusant utilizing PBI-3939 substrate hormone induction turns The biodiversity resources of position.
Figure 91 A-B shows the Protein kinase C α (PKC of the OgLuc fusant utilizing PBI-3939 substrate phorbol exters to induce α) the biodiversity resources of indexing.
Figure 92 A-B shows that the bioluminescence of the autophagosome protein translocation utilizing PBI-3939 substrate OgLuc fusant becomes Picture.
Describe in detail
Before any embodiment of the present invention explained in detail, it should be understood that ground is to the invention is not restricted to it about following The structure of the component shown in listed or the following drawings in description, the application synthesizing and arranging.The present invention is about specifically Embodiment and technology describe, but, the present invention may have other embodiments and may put into practice in a different manner Or carry out.
Unless otherwise indicated, in the following description of the method for the present invention, process step is at room temperature (about 22 DEG C) and air Carry out under pressure.Also it should be especially appreciated that ground is any digital scope listed by this paper includes owning from lower limit to higher limit Value.Such as, if the concentration range of indicating or beneficial effect range are 1% to 50%, it means the most clearly enumerates Such as 2% to 40%, 10% to 30% or 1% to 3% etc..Similarly, if given sequence homogeneity scope is, such as Between 60% to ＜ 100%, it means and the most clearly lists such as 65%, 75%, 85%, 90%, 95% Deng.These are only concrete indication why example, and think and clearly indicate from minimum to peak in this specification All possible numerical value.
Unless expressly indicated otherwise, the term in the literary composition of the application " includes " being intended to point out except being drawn by " including " List member outside may be optionally present other member.However, it is contemplated that term " includes " containing there is not other one-tenth The specific embodiment that probability is the present invention of member, i.e. the purpose of this embodiment is interpreted as having for will " include " " by ... composition " implication.
The following detailed description discloses the specific and/or preferred version of the single feature of the present invention.This Invention also contemplates as particularly preferred embodiment, and those embodiments are by by about two in inventive feature Or two or more combinations in multiple described specific and/or preferred version and generate.
Unless expressly indicated otherwise, all instructions of the relative quantity in the application generate based on w/w. The instruction of the relative quantity of the component characterized by generic term is intended to refer to all specific change comprised by described generic term Change form or the total quantity of member.If indicating and being existed with certain relative quantity by certain component defined in generic term, and if this component Also being characterized as being the specific version or member comprised by this generic term, it means and additionally there is not this generic term The other version comprised or member, thus this generic term total relative quantity of component of being comprised has exceeded and indicates Relative quantity.It is highly preferred that the other version that comprised of generic term or member are completely absent.
Summary
At different aspects, the present invention is about new compound, novel fluorescence element enzyme and a combination thereof.The present invention contains and includes Described new compound, novel fluorescence element enzyme and/or the method for a combination thereof, compositions and test kit.
Described new compound is novel coelenterazine, and it can be used as the substrate utilizing coelenteron usually to produce luminous albumen, Described albumen includes, but not limited to luciferase and the luminescent protein such as cnidaria found in different marine organisms Luciferase (such as, sea pansy belongs to luciferase), jellyfish luciferases are (such as, from many siphonohore (Aequorea Jellyfish) aequorin) (such as, the luciferase of thin Fructus Gleditsia acupuncture shrimp is combined for luciferase and decapods luciferase Thing).In different embodiments, the novel coelenterazine of the present invention has the physical stability of enhancing relative to natural coelenterazine (such as, the coelenterazine stability of enhancing), the self-luminous reduced and increase with the biocompatibility of cell (such as, to cell Less toxicity, including the cell type of allos) at least one.
Novel fluorescence element enzyme disclosed herein includes the variant of the active subunits of decapods luciferase.Novel fluorescence element Enzyme may utilize different coelenterazine as substrate, includes, but not limited to the new of natural and known coelenterazine and the present invention Type coelenterazine.Novel fluorescence element enzyme show following at least one: the luminescence of enhancing (brightness that includes increasing, enhancing Signal stabilization and/or signal duration)；(that is, the enzymatic activity of enhancing, including the temperature for raising for the enzyme stability strengthened The toleration of the enhancing of degree, the change of pH, inhibitor, denaturant and/or detergent)；The substrate specificity changed is (i.e., relatively The change of substrate specificity)；With strengthen biocompatibility (include the expression in the cell increased, the toxicity of reduction and/or At least one in cellular stress).In different embodiments, the present invention contains novel fluorescence element enzyme, described novel fluorescence Element enzyme as solvable activated monomer or is connected (such as, fusion protein) with other molecular chemistries and exists in solution, or attachment On a solid surface (such as, granule, capillary tube or detection pipe or flat board).
Some combination of novel coelenterazine and novel fluorescence element enzyme provides significant technological merit to bioluminescence assay, Including the luminescence strengthened, the luminescence wherein strengthened can be owing to including the coelenterazine stability of signal stabilization and the enhancing strengthened Caused by one or more factors.Additionally, a lot of novel coelenterazine are designed to less than that be obtained commercially and/or known coelenteron Element.In some cases, the novel fluorescence element enzyme of the present invention is preferably by being better than that be obtained commercially and/or known bigger Novel, the less coelenterazine of coelenterazine.
The present invention contains following combination: novel fluorescence element enzyme variants and novel coelenterazine；Novel fluorescence element enzyme variants with Known or natural coelenterazine；With novel coelenterazine and use coelenterazine as any of or natural albumen of substrate (such as luciferase or luminescent protein).
Term " coelenterazine " refer to naturally occurring (" natural ") coelenterazine with and the like, including coelenterazine-n, chamber Intestinal element-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, double deoxidation coelenteron Element (" coelenterazine-hh "), coelenterazine-i, coelenterazine-icp, coelenterazine-v and 2-methyl coelenterazine, and WO2003/040100 Disclosed those with in U. S. application the 12/056th, 073 ([0086] section), the disclosure of which is incorporated herein by reference. Term " coelenterazine " also refers to novel coelenterazine (see below) disclosed herein.Term " known coelenterazine " refers in the present invention The most known coelenterazine analog.
Term " OgLuc " refers to decapods luciferase protein, or the variant of this albumen, and it is raw in the presence of coelenterazine Cheng Guang.OgLuc albumen may be monomer or the subunit being probably albumen composition in its naturally occurring form.Institute is public herein OgLuc used in the exemplary embodiment opened is the 19kDa subunit of the luciferase complex from thin Fructus Gleditsia acupuncture shrimp, but Similar polypeptide from other decapods species (including that other sting shrimp species) is used as and is covered by the present invention (ginseng See R.D.Dennell, Observations on the luminescenceof bathypelagic Crustacea Decapoda of the Bermuda area, Zool.J.Linn.Soc., Lond.42 (1955), the 393-406 page；Also join See in JIUYUE, the 1999 .Inventaire document é des especies et bilan des formes les such as Poupin plus communesde la mer d′Iroise.Rapport Scientifique du Laboratoire d′Océ anographiedeNavale (LOEN), Brest (page 83), its each item is incorporated herein by reference)；Example bag Including but be not limited to, following luciferase: Xu Xia section (Aristeidae), including manomelia nearly prawn (Plesiopenaeus coruscans)；Pandalidea section, including Heterocarpus (Heterocarpus) and Parapandalus richardi, pipe Bian Xia section (Solenoceridae), including prawn (Mesopenaeus between Hymenopenaeus debilis and the torrid zone tropicalis)；Ying Xia section (Luciferidae), including eurymeric firefly shrimp (Lucifer typus)；Sergestidae (Sergestidae), including Atlantic Ocean rosy clouds shrimp (Sergestes atlanticus), arctic cherry shrimp (Sergestes Arcticus), point volume rosy clouds shrimp (Sergestes armatus), Sergestes pediformis, Sergestes Cornutus, Sergestes edwardsi, Sergestes henseni, comb rosy clouds shrimp (Sergestes pectinatus), Sergestes sargassi, the north rosy clouds shrimp (Sergestes similis), Sergestes vigilax, Sergiachallengeri, Sergia grandis, positive cherry shrimp (Sergia lucens), red cherry shrimp (Sergiaprehensilis), Sergia potens, Sergia robusta, flicker cherry shrimp (Sergia scintillans) With Sergia splendens；Pasiphaeidae (Pasiphaeidae), including Glyphusmarsupialis, the thin crayfish in Bermuda (Leptochela bermudensis), ditch volume Vulpes glass shrimp (Parapasiphae sulcatifrons) and Pasiphea tarda；Ci Xia section (Oplophoridae), including Acanthephyra acanthitelsonis, Acanthephyra Acutifrons, Acanthephyra brevirostris, Acanthephyra cucullata, short bending angle spine shrimp (Acanthephyra curtirostris), abnormal spine shrimp (Acanthephyra eximia), Acanthephyra gracilipes、Acanthephyra kingsleyi、Acanthephyra media、Acanthephyra Microphthalma, Acanthephyra pelagica, Acanthephyraprionota, purple thorn dish shrimp (Acanthephyra purpurea), blood red thorn dish shrimp (Acanthephyra sanguinea), Acanthephyra sibogae、Acanthephyrastylorostratis、Ephyrina bifida、Ephyrina figueirai、 Ephyrina koskynii, flat-shaped flat shrimp (Ephyrina ombango), cold water film first shrimp (Hymenodora Glacialis), thin cyst membrane shrimp (Hymenodora gracilis), Meningodora miccyla, soft leather bag shrimp (Meningodora mollis), Meningodora vesca, oncus back of a bow shrimp (Notostomusgibbosus), Notostomus auriculatus, thin Fructus Gleditsia acupuncture shrimp, Oplophorus grimaldii, Oplophorus novaezealandiae、Oplophorus spinicauda、Oplophorusfoliaceus、Oplophorus Spinosus, typical case's thorn shrimp (Oplophorus typus), Systellaspis braueri, Systellaspis Cristata, weak contracting first shrimp (Systellaspisdebilis) and sparkling and crystal-clear ventral spine shrimp (Systellaspis pellucida)； With Penaeus seu panulirus section (Thalassocaridae), intend green long volume shrimp (Chlorotocoides spinicauda) including thorn tail, by hair Penaeus seu panulirus (Thalassocaris crinita) and Thalassocaris lucida.
SEQ ID NO:1 gives (that is, signal-sequenceless) thin Fructus Gleditsia acupuncture shrimp luciferase (that is, BAB13776 of maturation The 28th 169 aminoacid to the 196th residue) the peptide sequence of 19kDa subunit of the form that naturally occurs.In difference Embodiment in, in OgLuc sequence (such as, if shown in C1+A4E peptide sequence SEQ ID NO:3) initial of synthesis Insert methionine residue and valine residue to aid in the clone in Heterologous System and expression.But, for concordance, herein The Position Number of the different amino acid replacement being previously mentioned " relative to " SEQ ID NO:1, i.e. ripe (signal-sequenceless), The peptide sequence of the natural 19kDa subunit of thin Fructus Gleditsia acupuncture shrimp luciferase protein complex illustrates.
Specifically, if albumen comparison based on its aminoacid sequence Yu SEQ ID NO:1, sequence iden is ＞ 30%, Preferably ＞ 40%, and most preferably ＞ 50%, then be decapods luciferase, and this albumen may utilize coelenterazine as substrate With the transmitting of catalytic luminescence, and the aminoacid sequence originating in the position of corresponding to SEQ ID NO:1 the 8th is:
[GSAIVK]-{FE}-[FYW]-x-[LIVMFSYQ]-x-x-{K}-x-[NHGK]-x-[DE]-x-[LIVMFY]- [LIVMWF]-x-{G}-[LIVMAKRG] (SEQ ID NO.330) (VII),
Have less than 5 differences, or no more than 4,3,2 or 1 difference, or most preferably indifference Different, wherein said difference occurs in the mode position 1,2,3,5,8,10,12,14,15,17 corresponding to the formula (VII) according to table 4 Or 18 position in.Difference may also include the room between the mode position of table 4 or insertion.
Term " variant " refers to the form of the modification of starting polypeptide or polynucleotide sequence.Term " parental generation " is relative terms, Refer to the most adorned homing sequence.Parental sequences common name is used as by the reference of the albumen coded by the sequence of the modification of gained, Such as, to compare activity level or other character of the albumen coded by the sequence by parental generation and modification.Homing sequence can be Naturally occurring (that is, natural or wild type) sequence.Homing sequence can also is that thus the variant sequence that is further embellished Row.When in the initial, middle of sequence and/or end displacement, lacking and/or add one or more aminoacid (it can be sky That so exist or synthesis) time, peptide sequence is " modification ".When in the initial, middle of sequence and/or end displacement, disappearance And/or when adding one or more nucleotide but maybe may will not change the aminoacid coded by sequence, polynucleotide sequence It is " modification ".In some embodiments, modification produces the functional fragment as specific OgLuc or OgLuc variant Variant.Functional fragment is that the parental sequences with total length has identical functional activity and is less than the sheet of the parental sequences of total length Section.Functional activity is the ability that performance is luminous.In some embodiments, modification produces the turnaround sequence as parental sequences Variant, such as circulates turnaround sequence and includes disappearance and/or the turnaround sequence inserted.
Some in OgLuc variant disclosed herein have been designated breviary name to assist to discuss.Term " C1+A4E " (also referred to as " C1A4E ") refer to relative to SEQ ID NO:1 have amino acid replacement A4E, Q11R, A33K, V44I, A54F, (wherein form " x#y " refers to be replaced by change the OgLuc variant (SEQ ID NO:2 and 3) of P115E, Q124K, Y138I and N166R The parent amino acid " x " of the " # " position of body aminoacid " y ").Except as otherwise noted, the C1+A4E OgLuc variant occurred herein Variant comprise at least amino acid replacement seeing C1+A4E.Term " IVY " refers to have other ammonia relative to SEQ ID NO:1 The variant (SEQ ID NO:8 and 9) of the C1+A4E OgLuc variant of base acid displacement F54I, I90V and F77Y.Term " IV " refers to phase SEQ ID NO:1 is had to another variant (SEQ of the C1+A4E OgLuc variant of other amino acid replacement F54I and I90V ID NO:14 and 15).Term " QC27 " refers to have other amino acid replacement Q18L, F54I, L92H relative to SEQ ID NO:1 Another other variant (SEQ ID NO:4 and 5) with the C1+A4E OgLuc variant of Y109F.Term " QC27-9a " refers to phase SEQ ID NO:1 is had to the QC27OgLuc of other amino acid replacement V21L, F68Y, L72Q, M75K, H92R and V158F The variant (SEQ ID NO:6 and 7) of variant.Term " 9B8 " refers to have other amino acid replacement relative to SEQ ID NO:1 The variant (SEQ ID NO:18 and 19) of the IV OgLuc variant of Q18L, F68Y, L72Q and M75K.Term " 9B8opt " refers to 9B8 The codon optimized form (SEQ ID NO:24) of variant.Term " 9B8opt+K33N " refers to have relative to SEQ ID NO:1 The variant (SEQ ID NO:42 and 43) of the 9B8opt variant of other amino acid replacement K33N.Term " 9B8opt+K33N+ 170G " refer to the glycine relative to SEQ ID NO:1 with the other C-end being additional to variant, the i.e. " 9B8opt+ of 170G K33N " variant (SEQ ID NO:68 and 69) of variant.Term " L27V+T39T+K43R+Y68D " and " L27V " refer to relative to SEQ ID NO:1 has the variant of " 9B8opt+K33N " variant of other amino acid replacement L27V, T39T, K43R and Y68D (SEQ ID NO:88 and 89).Term " T39T+K43R+Y68D " and " V2 " refer to have other ammonia relative to SEQ ID NO:1 The variant (SEQID NO:92 and 93) of " 9B8opt+K33N " variant of base acid displacement T39T, K43R and Y68D.
Common name, " enhancing " means and adds coelenterazine combination or the luciferase discussed relative to reference to luciferase, special Fixed character (such as, luminescence, signal stabilization, biocompatibility, protein stability (such as, enzyme stability) or protein expression) Increase, wherein said increase as adding coelenterazine combination or the luciferase discussed at least higher than described with reference to luciferase 1%, at least 5%, at least 10%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, at least 100%, At least 200%, at least 500% or at least 1000%.
Term " luminous " refers to the light output of OgLuc variant under suitable condition, such as, at suitable substrate such as coelenteron In the presence of element.Light output can be as the instantaneous of light output during luminescence-producing reaction initial or nearprompt measurement (sometimes referred to as " T =0 " luminescence or " flash of light ") to measure, it can start after adding coelenterazine substrate.In different embodiments, luminous anti- Should carry out in the solution.In further embodiment, luminescence-producing reaction is carried out on a solid support.Solution can comprise lysate, Such as from the cell in protokaryon or eukaryotic expression system.In further embodiment, express and occur at cell free system, or Luciferase protein is secreted in extracellular medium, thus, in the later case, it is not necessary to produce lysate.Some embodiment party In case, reaction is by by suitable raw material such as, and (such as, coelenterazine, buffer etc. are injected into the reative cell comprising luminescent protein The hole of porous plate such as 96 orifice plate) in initiate.In other other embodiments, OgLuc variant and/or novel coelenteron Element is directed in host and carries out the measurement of luminescence on host or its part, and described host or its part can include complete Organism or its cell, tissue, outer implant or extract.Reative cell can be located at and can such as utilize photometer or photomultiplier to survey In the reading plotter of amount light output.Also can elapse in time and measure light output or luminous, such as, hold in identical reative cell Continuous several seconds, the time period of a few minutes, several hours etc..Light output or the luminous recordable meansigma methods for a period of time, the declining of signal Half-life, the summation of the signal of a period of time or the highest output subtracted.Luminescence can be measured with relative light unit (RLU).
" luminescence of enhancing " of OgLuc variant can cause due to one or more in following characteristics: the light of enhancing is defeated Go out (that is, brightness), the substrate specificity, the signal stabilization of enhancing and/or the signal duration of enhancing that strengthen.The letter strengthened Number stability includes the increase of the time of the signal continuous illumination from luciferase, such as, as by the letter in time course Number decay half-life measured by.The luminescence strengthened can relative to natural, known or novel substrate is combined Luciferase such as wild type OgLuc, OgLuc misfolded proteins, sea pansy belong to luciferase (such as, hRluc) or Lampyridea fluorescence The comparable character of element enzyme (such as, from the Luc2 luciferase of North America Lampyridea (Photinus pyralis)) is come really Fixed, as shown in the following example.Such as, utilize in following example one or more in disclosed mensuration, can by with The luminescence of the given OgLuc variant that specific coelenterazine (including natural, known or novel coelenterazine) combines with this In any combination of OgLuc variant C1+A4E, IV or IVY natural, in known or novel coelenterazine disclosed in literary composition A kind of character compares.Especially, can be by measuring by bacterial lysate and the substrate PBI-containing discussed OgLuc variant The luminous signal (RLU) hatching generation of 3939 determines the luminescence of enhancing.Measure and may comprise TERGITOLTMReagent in Carrying out providing Glo sample kinetics, such as, enzyme inactivation slows down and luminous signal is stable wherein, and it is on other ground of the application Side is described.In some embodiments, some luciferase variants, such as, L27V, with some compound, such as, PBI- 3939, it is provided that the persistent period of the prolongation of luminescence emissions, or there is not TERGITOLTMTime aura sample kinetics occurs.Can Luminous signal and reference point such as C1+A4E variant and coelenterazine or coelenterazine-h or sea pansy are belonged to luciferase and natural coelenteron The luminous signal of element compares.
" enzyme stability " refers to the stability (that is, the enzymatic activity toleration to reaction condition) of enzymatic activity.The enzyme strengthened is stable Property refer to enzymatic activity enhancing stability (that is, to reaction condition strengthen toleration).The enzyme stability strengthened includes enhancing Heat stability (such as, stability at elevated temperatures) and chemical stability (such as, are such as gone at inhibitor or denaturant Dirt agent (includes such as TRITONTMX-100) stability in the presence of).Especially under conditions of known destruction protein structure, all In the presence of high temperature or chemical denaturant, enzyme stability can be used as measuring of protein stability.Especially, available such as the application In heat described by elsewhere (such as, in embodiment 28) analyze and determine the protein stability of enhancing.Luminescence can be believed Number compared with the reference point that C1+A4E variant and coelenterazine or coelenterazine-h or sea pansy belong to luciferase and natural coelenterazine.
" biocompatibility " phalangeal cell (such as, protokaryon or eucaryon) is to luciferase and/or coelenterazine compound Toleration.The biocompatibility of luciferase and/or coelenterazine with its cause on host cell stress be relevant.Such as, carefully The luciferase that born of the same parents do not tolerate may will not be expressed in cell effectively, and such as, this luciferase may be expressed in cell, But owing to the formation of the inclusion body of expressed albumen causes the activity that shows reduction.The biocompatibility of luciferase is with thin The ability of the tolerance of the insertion of exogenous gene is correlated with by born of the same parents, and described exogenous gene i.e., comprises coding fluorescence element enzyme or its fragment The transgenic of gene, have whereby the cell of transgenic will not show stress performance, described stress performance include stress (such as, the number of the living cells of reduction, the reduction of the viability of the induction of reaction path, the growth rate of reduction and/or reduction Film integrality or the apoptosis speed increased).Other instructions of cellular stress can include gene expression, signal pathway and/or regulation and control The change of approach.The biocompatibility of the enhancing of OgLuc variant can be due to the protein expression such as strengthened and/or the cell of reduction Stress factor caused.Luminous expression about the enhancing of the specific polynucleotide of coding OgLuc variant can be relative to The luminous expression of the polynucleotide (including codon optimized polynucleotide) of encoding wild type OgLuc or OgLuc misfolded proteins Level determines, luminescence activity wherein can be used as the instrument of monitoring protein expression level.
Especially, the biocompatibility of the enhancing of OgLuc variant, novel coelenterazine compound and/or a combination thereof can pass through The speed of growth measuring cell survival and/or cell determines.Such as, the biocompatibility of the enhancing of OgLuc variant can lead to Cross measure in the case of there is not any coelenterazine compound with comprise LUC Photinus pyralis LUC Photinus pyralis FL or sea pansy belong to luciferase or Do not comprise the growth rate of the cell survival comprising OgLuc variant that the cell of luciferase compares and/or cell with really The degree of the compatible of cell and/or toxicity is determined by this luciferase fixed.The biofacies of the enhancing of novel coelenterazine compound Capacitive can be by measuring compared with natural or known coelenterazine in the case of the luciferase expression that there is not cell It is exposed to the cell survival of novel coelenterazine compound to determine the compatibility of coelenterazine compound on intracellular and/or toxicity Degree determines.OgLuc variant can be comprised by measurement with the biocompatibility of the enhancing of the combination of novel coelenterazine compound OgLuc variant is also exposed to novel coelenterazine and comprises LUC Photinus pyralis LUC Photinus pyralis FL or sea pansy belongs to luciferase or do not conforms to luciferase And the speed of growth of the cell survival that compares of the cell being exposed to natural or known coelenterazine and/or cell comes really Fixed.
Especially, the biocompatibility of enhancing available as in the application elsewhere described by cell survival divide Analysis (such as, utilizes as described in embodiment 18Measure or such as saying according to manufacturer Bright utilizationThe apoptosis of technology measures) or analysis as known in the art determine.OgLuc variant pair The effect of cell survival or apoptosis can belong to glimmering with reference to luciferase such as C1+A4E variant, LUC Photinus pyralis LUC Photinus pyralis FL or sea pansy The effect of light element enzyme compares.The effect of novel coelenterazine compound on intracellular viability or apoptosis can be with natural or known The effect of coelenterazine compound on intracellular viability or apoptosis compares.
The biocompatibility strengthened can by measure OgLuc variant and/or novel coelenterazine compounds on cell growth or The effect of gene expression determines.Such as, the biocompatibility of the enhancing of OgLuc variant can be thin by measuring over time Born of the same parents' number determine or stress response gene in the sample of cell that is determined by comprising OgLuc variant expression with comprise another One luciferase or the cell without luciferase are compared and are relatively determined.The biocompatibility of the enhancing of novel coelenterazine compound Can determine or be exposed to by mensuration the cell of novel coelenterazine compound by measuring cell number over time The expressing and be exposed to natural or known coelenterazine or be not exposed to the cell of coelenterazine of stress response gene in sample Compare and relatively determine.The effect of OgLuc variant cell growth or gene expression can be with reference to luciferase, and such as C1+A4E becomes Body, LUC Photinus pyralis LUC Photinus pyralis FL or sea pansy belong to luciferase and compare.Novel coelenterazine cell growth or the effect of gene expression Can be compared with natural or known coelenterazine.
The bright signal of luciferase and the small size of OgLuc gene can beneficially identify the Cytoplasm expressing the present invention Firm, the stable cell line of the OgLuc variant of form or secreted form.Expect that relatively small gene order will reduce by outward Source DNA is incorporated in cellular genome the probability of the genic instability produced.The most natural with other known luciferases OgLuc, LUC Photinus pyralis LUC Photinus pyralis FL or sea pansy belong to luciferase and compare, due to OgLuc variant and/or the novel cavity of the present invention The brightness of increase of intestinal element, less protein expression and thus less DNA needed for transfection can produce the brightness of given level, It contributes to the biocompatibility of enhancing of OgLuc variant and/or novel coelenterazine.The bio-compatible of the enhancing of OgLuc variant Property can by transient transfection generates have the OgLuc the most natural with utilizing other luciferase, LUC Photinus pyralis LUC Photinus pyralis FL or DNA or the amount of reagent needed for the cell of the luminescence that sea pansy belongs to the cell phase same level that luciferase is transfected are measured, described Reagent such as transfects chemical substance.In some embodiments, obtain with utilizing other luciferase to generate to have for transfection The identical level of luminescence transfection cell needed for OgLuc modification D NA or the amount of reagent less than another luciferase example As natural OgLuc, LUC Photinus pyralis LUC Photinus pyralis FL or sea pansy belong to the amount needed for luciferase.The biofacies of the enhancing of OgLuc variant Capacitive can be measured by the recovery time of Transfected cells.In some embodiments, after utilizing the transfection of OgLuc variant OgLuc, LUC Photinus pyralis LUC Photinus pyralis FL or sea pansy that the amount of the time needed for recovery is less than another luciferase the most natural belong to fluorescence Time needed for element enzyme.
" relative substrate specificities " is divided by with reference to coelenteron by the luminescence of luciferase in the presence of inspection coelenterazine substrate In the presence of element, the luminescence of luciferase determines.Such as, relative specificity can be by utilizing the novel coelenterazine of the present invention The luminescence of luciferase divided by utilize different coelenterazine (such as, natural or known coelenterazine, see Fig. 1 about example, Or the different novel coelenterazine of the present invention) the luminescence of luciferase determine.Inspection coelenterazine substrate and being compared It is considered as the comparison substrate pair for determining relative substrate specificities with reference to coelenterazine substrate.
It is special that " change of relative substrate specificities " compares the substrate relative substrate to checking luciferase by utilization Property determines divided by the relative substrate specificities of the reference luciferase utilizing identical comparison substrate pair.Such as, the most special The change of property can by from different coelenterazine (such as, the different novel cavity of natural or known coelenterazine or the present invention Intestinal element) compare utilize the present invention novel coelenterazine inspection luciferase relative substrate specificities divided by with for checking The identical different coelenterazine of luciferase compares reference luciferase relative of the novel coelenterazine utilizing the present invention Substrate specificity.
In some embodiments, the luminescence of a kind of novel coelenterazine and the luminescence utilizing different novel coelenterazine are utilized Compare.In some embodiments, utilize the luminescence of a kind of natural or known coelenterazine with utilize another natural or The luminescence of known coelenterazine compares.In other other embodiments, utilize a kind of natural or known coelenteron The luminescence of element is compared with the luminescence utilizing novel coelenterazine.
The novel coelenterazine of the present invention include the physical stability (such as, the coelenterazine stability of enhancing) that such as strengthens or The self luminous character reduced.The physical stability of coelenterazine refers to that the degree of stability of coelenterazine under certain conditions makes when being used as During the substrate of luciferase, it maintains luminous ability.The luminescence of the activity not relying on luciferase or luminescent protein is referred to as certainly Luminous.Self-luminous is the luminescence of the material produced by the energy discharged in the form of light in decay or catabolic process.Such as, certainly Luminescence can cause due to the spontaneous oxidation of luminogen substrate coelenterazine.
As used herein, " pure " or " purification " mean object material be existing main matter (that is, with mole And/or based on quality, in addition to water, solvent, buffer or other usual ingredients of the aqueous system in compositions, its ratio is any Other single materials are more rich), and, in some embodiments, the fraction of purification is that wherein object material accounts for existing All macromolecular substances at least about 50% (by mole based on) compositions.Generally, " the purest " compositions will account for Present in compositions about more than the 80% of every other macromolecular substances, in some embodiments, about more than 85%, about More than 90%, about more than 95% or about more than 99%.In some embodiments, object material is purified to substantially homogeneity (impurity substances in compositions can not be detected by common detection methods), wherein compositions is substantially by single macromole Material is formed.
Coelenterazine derivant
In some embodiments, the invention provides the novel coelenterazine derivant of formula (Ia) or (Ib):
Or
Wherein R2Group selected from consisting of:
Or C2-5Directly Alkyl group；
R6Choosing freely-H ,-OH ,-NH2,-OC (O) R or-OCH2The group of OC (O) R composition；
R8Choosing is freelyH or the group of low-grade cycloalkyl composition；
Wherein R3And R4It is H or is C1-2Alkyl；
W is-NH2, halo ,-OH ,-NHC (O) R ,-CO2R；
X is-S-,-O-or-NR22-；
Y is-H ,-OH or-OR11；
Z is-CH or-N-；
Each R11Independently be-C (O) R " or-CH2OC(O)R”；
R22For H, CH3Or CH2CH3；
Each R independently be C1-7Straight chained alkyl or C1-7Branched alkyl；
R " it is C1-7Straight chained alkyl or C1-7Branched alkyl；
The key table of dotted line shows the existence of optional ring, and it can be saturated or unsaturated；
Precondition is to work as R2ForOrTime, R8It is not
Precondition is to work as R2ForTime, R8It isOr low-grade cycloalkyl；With
Precondition is to work as R6For NH2Time, R2ForOr C2-5Alkyl；
Or R8It is not
As used herein, term " alkyl " belongs to the unit price portion obtained by taking out hydrogen atom from hydrocarbon compound Dividing, and it can be saturated, part is undersaturated or the most undersaturated.Alkyl can be straight chain or side chain.Alkyl Can optionally be replaced by such as halo.The example of straight chained alkyl include, but not limited to ethyl, n-propyl, n-butyl and N-propyl, n-hexyl and n-heptyl.The example of the undersaturated alkyl with one or more carbon-to-carbon double bond includes, but not It is limited to, vinyl (vinyl ,-CH=CH2), 2-acrylic (pi-allyl ,-CH-CH=CH2), and cyclobutenyl.Have one or The example of the undersaturated alkyl of multiple carbon-to-carbon triple bonds includes, but not limited to acetenyl and 2-propynyl (propargyl).Side chain The example of alkyl includes isopropyl, isobutyl group, sec-butyl, the tert-butyl group and isopentyl.
As used herein, term " low-grade cycloalkyl " belongs to by from the hydrocarbon compound with 3 to 5 carbon atoms Dehydrogenation atom and the monovalent moiety that obtains.The example of saturated low-grade cycloalkyl include, but not limited to group such as cyclopropyl, Cyclobutyl and cyclopenta.The example of the undersaturated low-grade cycloalkyl with one or more carbon-to-carbon double bond includes, but does not limits In, group such as cyclopropanyl, cyclobutane base and cyclopentenyl.
As used herein, term " halo " belongs to halogen, such as Cl, F, Br or I.
In some embodiments, R2It isAnd X is O or S.In further embodiment, R2It is C2-5Straight chained alkyl.In certain embodiments, R8It isLow-grade cycloalkyl or H.In further embodiment, R8It it is benzyl.In some embodiments, R " it is-C (CH3)3、-CH(CH3)2、-CH2C(CH3)3Or-CH2CH(CH3)2。
In some embodiments, the invention provides according to formula (IIa) or the compound of (IIb):
Or
Wherein X is O or S, R6It is H or OH, R11As defined above, and the key table of dotted line shows the existence of optional ring.
In some embodiments, the invention provides formula (IIIa) or the compound of (IIIb):
Or
Wherein R12It is C2-5Straight chained alkyl, furyl or thienyl, R6It is H or OH, R11As defined above, and dotted line Key table shows the existence of optional ring.
In some embodiments, the invention provides formula (IVa) or the compound of (IVb):
Or
Wherein X is O or S, R6It is H or OH, R8Be H,Or low-grade cycloalkyl, R3、R4And R11Such as above institute Define, and the key table of dotted line shows the existence of optional ring.
In some embodiments, the invention provides according to formula (Va) or the compound of (Vb):
Or
Wherein R8It is benzyl, R11As defined above, and the key table of dotted line shows the existence of optional ring.
In some embodiments, the invention provides the novel coelenterazine derivant of formula (VIa) or (VIb):
Or
Wherein R2Choosing is freely Or C2-5The group of straight chained alkyl composition；
R6Choosing freely-H ,-OH ,-NH2,-OC (O) R or-OCH2The group of OC (O) R composition；
R8Group selected from consisting of:
H or low-grade cycloalkyl；
Wherein R3And R4It is H or is C1-2Alkyl；
W is-NH2, halo ,-OH ,-NHC (O) R ,-CO2R；
X is-S-,-O-or-NH-；
Y is-H ,-OH or-OR11；
Z is-CH or-N-；
Each R11Independently be-C (O) R " or-CH2OC(O)R”；
Each R independently be C1-7Straight chained alkyl or C1-7Branched alkyl；
R " it is C1-7Straight chained alkyl or C1-7Branched alkyl；
The key table of dotted line shows the existence of optional ring, and it can be saturated or unsaturated；
Precondition is to work as R2ForOr Time, R8It is not
Precondition is to work as R2ForTime, R8It isOr low-grade cycloalkyl；With
Precondition is to work as R6For NH2Time, R2ForOr C2-5Alkyl；
Or R8It is not
Suitable compound according to the present invention includes
Isomer, salt and shielded form
Some compound can with one or more specific geometry, optics, enantiotopic, diastereoisomeric, Presented in epimerism, stereomeric, tautomeric, conformation or different head, include but not limited to, cis and anti- Formula；E type and Z-type；C type, t type and r type；Internally-oriented and export-oriented；R type, S type and meso-form；D type and L-type；D type and l type； (+) type and (-) type；Keto-acid, enol form and enol form；Cis and trans；To oblique formula and anticline form；Alpha form and beta form； Axially form and calm form；Boat form, chair form, torsion formula, envelope type and half-chair；And a combination thereof, hereinafter collectively referred to as " isomer " (or " isomeric form ").
Note, except discussed below in relation to tautomeric form, be particularly intended to exclude at " isomery as the term is employed herein Body " outside, for structure (or structure) isomer (that is, between atom to connect different rather than only atom locus different Isomer).Such as, methoxyl group ,-OCH are mentioned3, it is not necessarily to be construed as mentioning its constitutional isomer, methylol ,-CH2OH.Phase As, mention Chloro-O-Phenyl, be not necessarily to be construed as mentioning its constitutional isomer, a chlorphenyl.But, mention that a class formation can be complete Entirely include isomeric form (such as, the C belonging in such structure1-7Alkyl includes n-pro-pyl and isopropyl；Butyl includes positive fourth Base, isobutyl group, sec-butyl and the tert-butyl group；Anisyl includes guaiacyl, m-methoxyphenyl and p-methoxyphenyl).
Note, particularly in term " isomer " is to have the substituted compound of one or more isotope.Example As, H can be any isotope form, including1H、2H (D) and3H(T)；C can be any isotope form, including12C、13C With14C；O can be any isotope form, including16O and18O；With like this.
Unless otherwise indicated, mention that specific compound includes all such isomeric form, including (fully or part Ground) its racemic modification and other mixture.For preparing (such as asymmetric synthesis) and separating (such as fractional crystallization and chromatograph side Method) method of such isomeric form is as known in the art or by changing method teaching herein in known manner Or known method is easily obtained.
Unless otherwise indicated, mention that specific compound also includes its ion, salt, solvate and shielded form, Such as, as discussed below.The corresponding salt of preparation, purification and/or operation reactive compound can be convenient or preferable , such as, pharmaceutically acceptable salt.The example of pharmaceutically acceptable salt is at Berge etc., J.Pharm.Sci., 66:1-19 (1977) come into question in.
Such as, if compound is anion, or have can be anion functional group (such as ,-COOH can be- COO-), then may utilize suitable cation to form salt.The example of suitable inorganic cation includes, but not limited to alkali metal Ion such as Na+And K+, alkaline earth cation such as Ca2+And Mg2+, and other aniones such as Al3+.Suitable organic cation Example includes, but not limited to ammonium ion (that is, NH4+) and substituted ammonium ion (such as, NH3R+、NH2R2 +、NHR3 +、NR4 +).One The example of the most suitable substituted ammonium ion is derived from following those: ethamine, diethylamine, hexanamine, triethylamine, fourth Amine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, benzylaniline, choline, meglumine and tromethane, and amino Acid, such as lysine and arginine.The example of common quaternary ammonium is N (CH3)4 +。
If compound is cation, or to have can be functional group (the such as ,-NH of cation2Can be-NH3 +), then Available suitable anion forms salt.The example of suitable inorganic anion includes, but not limited to derive from following inorganic Those of acid: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulphuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid and phosphorous acid.Suitable organic the moon The example of ion includes, but not limited to derive from those of following organic acid: acetic acid, propanoic acid, succinic acid, glycolic, tristearin Acid, Palmic acid, lactic acid, malic acid, pamoic acid, tartaric acid, citric acid, gluconic acid, ascorbic acid, maleic acid, hydroxyl Malaysia Acid, phenylacetic acid, glutamic acid, aspartic acid, benzoic acid, cinnamic acid, acetone acid, salicylic acid, sulfanilic acid, 2-acetyloxy phenyl first Acid, fumaric acid, benzenesulfonic acid, p-methyl benzenesulfonic acid, methanesulfonic acid, ethyl sulfonic acid, ethane disulfonic acid, oxalic acid, pantothenic acid, isethionic acid, valeric acid, Lactobionic acid and gluconic acid.The example of suitable polymer anion includes, but not limited to come from that of following polymeric acid A little: tannic acid, carboxymethyl cellulose.
The corresponding salt of preparation, purification and/or operation reactive compound is probably convenient or preferable.As used herein Term " solvate " at its general sense middle finger solute (such as, the salt of reactive compound, reactive compound) and solvent Complex.If solvent is water, then solvent can be eligibly referred to as hydrate, such as, monohydrate, dihydrate, three hydrations Thing etc..
The reactive compound of preparation, purification and operating chemical forms of protection can be convenient or preferable.Such as this paper institute Term " chemoproection form " belong to wherein one or more reactive functional groups to be protected from undesirable chemistry anti- The compound answered, i.e. for blocking group (also referred to as sheltering group or blocking groups).By protection reactive functional groups, can enter Row relates to the reaction of other unprotected reactive functional groups, and does not affect blocking group；Generally can in a subsequent step by Blocking group is removed, and has no substantial effect on remaining part of molecule.See, e.g., Protective Groups in Organic Synthesis (T.Green and P.Wuts, Wiley, 1999).
Such as, hydroxyl can be shielded, as ether (-OR) or ester (-OC (=O) R), such as, as: the tert-butyl group Ether；Benzyl oxide, benzhydryl (diphenyl methyl) ether or trityl (trityl group) ether；Trimethylsilyl ethers or tertbutyl methyl Silica-based ether；Or acetonyl ester (-OC (=O) CH3,-OAc).Such as, aldehyde radical or ketone group can be protected respectively as acetal or ketal Protect, wherein carbonyl (＞ C=O) is converted into diether (＞ C (OR) by reacting with such as primary alconol2).Aldehyde radical or ketone group are prone to Regenerate by utilizing the water of excess to be hydrolyzed in the presence of acid.Such as, amido can be as such as amide or urethane Ester is protected, such as, as: methyl nitrosourea (-NHCO-CH3)；Benzyloxy-amide (-NHCO-OCH2C6H5,-NHCbz)；Make For tert-butoxy amide (-NHCO-OC (CH3)3,-NH-Boc)；2-xenyl-2-propoxyl group amide (-NHCO-OC (CH3)2C6H4C6H5,-NH-Bpoc), as 9-fluorenyl methoxy amide (-NH-Fmoc), as 6-nitro veratryl epoxide amide (- NH-Nvoc), as 2-trimethyl silicane ethyoxyl amide (-NH-Teoc), as 2,2,2-tri-chloroethoxy base amide (-NH- Troc), as allyloxy amide (-NH-Alloc), as 2-(phenyl sulfonyl) ethyoxyl amide (-NH-Psec)；Or, In a suitable manner, as N-oxide.
Such as, hydroxy-acid group can be by protection as ester, such as, as: C1-7Arrcostab (such as, methyl ester；The tert-butyl group Ester)；C1-7Haloalkyl ester (such as, C1-7Tri haloalkyl ester)；Three C1-7Alkyl tin groups, alkyl silane groups-C1-7Arrcostab；Or C5-20Aryl- C1-7Arrcostab (such as, benzyl ester；Nitrobenzyl ester)；Or as amide, such as, as methyl nitrosourea.
Such as, mercapto can be by protection as thioether (-SR), such as, as: benzyl thioether；Acetamidomethyl ether (- S-CH2NHC (=O) CH3)。
The synthesis of coelenterazine derivant
Coelenterazine derivant according to the present invention can synthesize according to those methods described in detail in embodiment 1-16.
Mutant thorn shrimp belongs to luciferase
In embodiments of the invention, multiple technologies described herein are employed to identify the site of amino acid replacement To produce the OgLuc polypeptide of the synthesis of improvement.Employ other technology with the polynucleotide of the different polypeptide of Optimized Coding Based Codon is to strengthen the expression of polypeptide.It is found that the amino acid replacement making one or more single or different combinations, produce Give birth to luminescence (such as, the brightness of enhancing, the signal stabilization of enhancing, the enzyme stability of enhancing and/or the relative end with enhancing The specific change of thing) the OgLuc type polypeptide of synthesis.Additionally, encode many nucleoside of the OgLuc variant polypeptide of different synthesis Acid includes that one or more codon optimized displacement creates the increasing of polypeptide in different eucaryons and prokaryotic expression system Strong expression.One embodiment of the invention is the polynucleotide of the OgLuc variant polypeptide of coding synthesis, as described OgLuc Variant polypeptide is solvable when expressing in protokaryon and/or eukaryotic cell and is active when monomeric form.
The OgLuc variant of the present invention can be with any albumen interested or molecule phase coupling interested.Implement at some In scheme, variant is fusion protein, such as, some variants be attached to N-end or C-endPolypeptide is even Connection.Unless otherwise noted, asThe variant of fusant includes " HT7 " part as its title, such as, “IVY-HT7”.In some embodiments, signal sequence (such as, naturally occurring thin Fructus Gleditsia acupuncture shrimp belongs to signal sequence) is attached to The N-end of fusion protein is secreted from cell with assistance fusion protein.Signal sequence, rather than OgLuc luciferase naturally occurs Signal sequence, be as known in the art to assist the protein excretion in mammalian cell or other cell types.Signal Sequence can be used for making OgLuc variant position on the outer surface of cell membrane or show with the combination of film anchor series.In this area Known additive method can also be used for other positions being positioned in film or cell by OgLuc variant.
In some embodiments, the invention provides the decapods luciferase of modification, it is relative to corresponding parental generation Variant decapods luciferase has the luminescence of enhancing.Such as, the variant OgLuc of parental generation be C1+A4E, IVY, IV, QC27, QC27-9a, 9B8,9B8opt+K33N, 9B8opt+K33N+170G, V2 or " L27V ".In another embodiment, the present invention Provide the decapods luciferase of the modification utilizing novel coelenterazine.In one embodiment, the decapods fluorescence of modification Element enzyme is for change natural, that known or novel coelenterazine has relative specificity.In one embodiment, modification Decapods luciferase has relative specificity relative to the variant decapods luciferase of corresponding parental generation and changes.
In some embodiments, the variant decapods luciferase of corresponding parental generation is decapods species, including from The different species of the section of Decapoda, include, but not limited to following luciferase: Xu Xia section (Aristeidae), including short The nearly prawn of limb (Plesiopenaeuscoruscans)；Pandalidea section, including Heterocarpus (Heterocarpus) and Parapandalus richardi, Guan Bian shrimp section (Solenoceridae), including Hymenopenaeusdebilis and the torrid zone Between prawn (Mesopenaeus tropicalis)；Ying Xia section (Luciferidae), including eurymeric firefly shrimp (Lucifer typus)；Sergestidae (Sergestidae), including Atlantic Ocean rosy clouds shrimp (Sergestes atlanticus), arctic cherry shrimp (Sergestes arcticus), point volume rosy clouds shrimp (Sergestesarmatus), Sergestes pediformis, Sergestes cornutus, Sergestes edwardsi, Sergestes henseni, comb rosy clouds shrimp (Sergestes Pectinatus), Sergestes sargassi, the north rosy clouds shrimp (Sergestes similis), Sergestes vigilax, Sergia challengeri, Sergiagrandis, positive cherry shrimp (Sergia lucens), red cherry shrimp (Sergia Prehensilis), Sergiapotens, Sergia robusta, flicker cherry shrimp (Sergia scintillans) and Sergia splendens；Pasiphaeidae (Pasiphaeidae), including Glyphus marsupialis, the thin crayfish in Bermuda (Leptochela bermudensis), ditch volume Vulpes glass shrimp (Parapasiphae sulcatifrons) and Pasiphea tarda；Ci Xia section (Oplophoridae), including Acanthephyraacanthitelsonis, Acanthephyra Acutifrons, Acanthephyra brevirostris, Acanthephyra cucullata, short bending angle spine shrimp (Acanthephyra curtirostris), abnormal spine shrimp (Acanthephyra eximia), Acanthephyra gracilipes、Acanthephyrakingsleyi、Acanthephyra media、Acanthephyra Microphthalma, Acanthephyra pelagica, Acanthephyra prionota, purple thorn dish shrimp (Acanthephyra purpurea), blood red thorn dish shrimp (Acanthephyra sanguinea), Acanthephyra sibogae、Acanthephyra stylorostratis、Ephyrina bifida、Ephyrina figueirai、 Ephyrina koskynii, flat-shaped flat shrimp (Ephyrina ombango), cold water film first shrimp (Hymenodora Glacialis), thin cyst membrane shrimp (Hymenodora gracilis), Meningodora miccyla, soft leather bag shrimp (Meningodora mollis), Meningodoravesca, oncus back of a bow shrimp (Notostomus gibbosus), Notostomus auriculatus, thin Fructus Gleditsia acupuncture shrimp, Oplophorus grimaldii, Oplophorus novaezealandiae、Oplophorus spinicauda、Oplophorus foliaceus、Oplophorus Spinosus, typical case's thorn shrimp (Oplophorus typus), Systellaspis braueri, Systellaspis Cristata, weak contracting first shrimp (Systellaspis debilis) and sparkling and crystal-clear ventral spine shrimp (Systellaspis pellucida)；With Penaeus seu panulirus section (Thalassocaridae), intend green long volume shrimp including thorn tail (Chlorotocoidesspinicauda), by hair Penaeus seu panulirus (Thalassocaris crinita) and Thalassocaris lucida.In some embodiments, the luciferase modified in prokaryotic cell and/or eukaryotic cell is relative to corresponding open country Raw type luciferase has a luminescence emissions increased, such as, at least 1.3 times, at least 2 times or at least 4 times.In some embodiments In, by one or more character of decapods luciferase modified and luciferase (the such as Lampyridea from another species Luciferase or sea pansy belong to luciferase) similar character compare.
In some embodiments, OgLuc variant and SEQ ID NO:3,5,7,9,11,13,15,17,19,21,27, 35、37、39、41、43、45、47、49、51、53、56、58、60、62、64、69、71、73、75、77、79、81、83、85、87、 89,91,93 or 95 have at least 60%, such as, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or the amino acid sequence identity of 99% or 100%.In some embodiments, OgLuc variant or its functional sheet Section, has less than 5 differences, or it is highly preferred that be less than 4,3,2 or 1 difference, or most preferably zero difference, Wherein said difference occur corresponding to the mode position 1,2,3,5,8,10,12,14,15,17 according to the formula (VII) of table 4 or In the position of 18.Difference may also include the room between the mode position of table 4 or insertion.
In some embodiments, the OgLuc variant of the present invention has one or more at N-end, C-end or two ends The aminoacid sequence of the allos fused polypeptide of epi-position or fusion tag (fused polypeptide such as have), it is optionally with interested Molecule either directly or indirectly interacts.In some embodiments, with interaction of molecules interested before or it After, the existence of heterologous sequence not substantially changes the luminescence of OgLuc variant.In some embodiments, heterologous amino acid sequence It it is epitope tag.In some embodiments, heterologous amino acid sequence is such, with the interaction of molecules phase interested Between or afterwards, experienced by conformational change, itself so that change the activity of OgLuc variant, such as there is such aminoacid sequence OgLuc variant for detection allosteric interaction be useful.OgLuc variant or with the fusant of OgLuc variant or its sheet Section can be used as report.
In some embodiments, the fragment of the OgLuc variant of the present invention and heterologous amino acid sequence merge, merge thus Forming β-bucket, this fusion protein can generate luminescence, its analog bag described from naturally occurring coelenterazine or its analog Include multiple known coelenterazine discussed in this article, or the novel coelenterazine of the present invention.
Also provide for be code book invention OgLuc variant or the polynucleotide of its fusant, have these polynucleotide or The host cell of the separation of OgLuc variant or its fusant, and use the polynucleotide of the present invention, OgLuc variant or its fusion Body or the method for host cell.
In the case of two or more nucleic acid or peptide sequence, term " homogeneity " refers to when utilizing any number of sequence Comparison algorithm or by manpower comparing to and macroscopic examination measured by leap comparison window or specify region about most homogeneous When comparing with comparison, identical or that there is specific percentage ratio identical amino acid residue or two of nucleotide or many Individual series or subsequence.The comparison method of the sequence for comparing is well known in the art.Optimal for the sequence that compares Comparison can be carried out by following: Smith etc., the algorithm of (J.Mol.Biol.147:195-197 (1981)), Needleman and Wunsch, the homology alignment algorithm of (J.Mol.Biol., 48:443-453 (1970)), Pearson and Lipman, The search for similarity method of (Proc.Natl.Acad.Sci.USA, 85:2444-2448 (1988)), algorithm computerized Implementing, such as, FASTA, SSEARCH, GGSEARCH (can service at the FASTA of University of Virginia William R.Pearson Device obtains http://fasta.bioch.virginia.edu/fasta_www2/fasta_intro.shtml), Clustal system Sequencer program (Chenna etc., Nucl.Acids Res.31 (13): 3497-3500 (2003)；Obtainable example is at http: // Www.ebi.ac.uk or http://www.ch.embnet.org), or other sequence analysis softwares.It is known in the art that Making of professional method is may relate to most homogeneous generation comparison having between the peptide sequence that significant sequence changes With, such as ABA method (Raphael etc., Genome Res.14 (11): 2336-2346 (2004)), other suitable methods, or Two the identical copies linked utilizing peptide sequence are compared.
" nucleic acid molecules ", " polynucleotide " or " nucleotide sequence " refers to nucleic acid as the term is employed herein, including DNA or RNA, It includes producing the code efficiency required for polypeptide or amyloid protein precursor.Coded polypeptide can be the polypeptide of total length, its fragment (less than total length), or the fusant of the polypeptide of total length or its fragment and another polypeptide, the fused polypeptide of generation.
The polynucleotide of encoding proteins or polypeptide mean the nucleotide sequence of the coding region including gene, or in other words, compile The nucleotide sequence of code gene outcome.Coding region can exist with cDNA, genomic DNA or rna form.When existing with DNA form Time, oligonucleotide can be strand (such as, sense strand) or double-strand.If desired, suitable control element such as enhancer/ Promoter, splice junction, polyadenylation signal etc. can be located at the coding region of contiguous gene to allow turning of primary RNA transcript thing Record and/or the appropriate of correct processing initiate.Other control or controlling element include, but not limited to Binding site for transcription factor, Splicing signal, polyadenylation signal, termination signal and enhancer element.
" peptide ", " albumen " and " polypeptide " means the amino acid chain of different length, regardless of whether post translational modification how (example As, glycosylation or phosphorylation).(that is, synthesis) misfolded proteins that the nucleic acid molecule encoding of the present invention is artificial or its polypeptide fragment Variant, its aminoacid sequence with its parent protein originated has at least 60%, such as, at least 65%, 70%, 75%, the amino acid sequence identity of 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, described parent (the natural or wild type) sequence can being naturally-occurring for albumen or the variant sequence thereof modified further subsequently.Term " fused polypeptide " or " fusion protein " refers to be included in N-end and/or C-end is (the most non-with one or more heterologous sequences OgLuc polypeptide) chimeric protein of reference protein (such as, OgLuc variant) that connects.Heterologous sequence can include, but not limited to report Accuse sub-albumen such asFusion protein (Promega Corp.), FlAsH (fluorescein spiral bonding agent Han arsenic) With ReAsH (the red spiral bonding agent Han arsenic) (such as, LUMIOTMTag recognition sequence (Invitrogen)), chloramphenicol acetyl turns Move enzyme (CAT), beta galactosidase (β-Gal), lactamase (P-gal), neomycin resistance (Neo), GUS, galactopyranose, Green fluorescent protein (GFP), luciferase (such as, sea pansy (Reniliareniformis) luciferase, firefly luciferin Enzyme (such as, North America Lampyridea (Photinuspyralis) or Photuris pennsylvanica) or Pleonomus luciferase (such as, Jamaica's Pleonomus (Pyrophorus plagiophthalamus) or Pyrearinus termitilluminans) Or Lampyridea (glowworm) luciferase (such as, Phrixothrix hirtus), xylosidase, thymidine kinase, Arab Glycosidase and SNAP label, CLIP label, ACP label and MCP label (New England Biolabs).An embodiment party In case, chimeric protein be included in N-end withThe OgLuc that fusion protein (Promega Corp.) connects becomes Body.In another embodiment, chimeric protein be included in C-end withThe OgLuc that fusion protein connects Variant.
Known nucleic acid comprises different types of " sudden change ", refer to relative to wild-type sequence in the sequence of nucleotide specific Change at base positions.Sudden change also can refer to the insertion of one or more base or disappearance so that nucleotide sequence is different from reference, Such as, wild-type sequence, or substitute by termination codon." replace " and refer to the amino acid whose change of specific location in sequence, Such as, E is become at the 4th from A.
Term " carrier " refers to wherein can be inserted into or clone the nucleic acid molecules of the fragment of DNA, and it can be used for shifting region of DNA section In cell and can replicate in cell.Carrier can derive from plasmid, phage, virus, cosmid and similar carrier.
As used herein, term " wild type " or " natural " refer to have the gene separated from naturally occurring source or The gene of the characteristic of gene outcome or gene outcome.Wild type gene is to be most frequently observed with in crowd and thus by arbitrarily " wild type " form of named gene.On the contrary, term " mutant " refers to when compared with wild type gene or gene outcome Functional character in the sequence and/or shows gene or the gene outcome of modification (that is, the characteristic of change).It is to be noted that it is natural The mutant existed can be separated；These are by it has the characteristic of change when compared with wild type gene or gene outcome The fact identified.
Exemplary polynucleotide and albumen
The present invention includes OgLuc variant or its albumen relative to wild type OgLuc with at least one amino acid replacement Fragment, such as, has disappearance, those of the disappearance of such as 1 to about 5 residue, and its chimera (fusant) (sees the U.S. special Profit is announced No. 2009/0253131 and WIPO and is announced No. WO2007/120522, and the disclosure of which is incorporated by reference into this Literary composition), this displacement causes OgLuc variant to have a luminescence of the stability of enhancing, enhancing, such as, the luminescence emissions of increase, higher Luminescence kinetics stability and/or the glow color of change.The amino of the sequence of OgLuc variant and corresponding wild type OgLuc Acid sequence is substantially the same.Polypeptide or the peptide with substantially the same sequence mean aminoacid sequence largely, but Not fully, identical and maintain the functional activity of associated sequence.Generally, if two aminoacid sequences its be at least 60%, such as, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%, but be less than The amino acid sequence identity of 100% is the most substantially the same.In some embodiments, OgLuc variant is by the many nucleoside recombinated Acid is coded.In some embodiments, OgLuc variant, or its function fragment, have less than 5 differences, or more preferably Less than 4,3,2 or 1 difference, or most preferably zero difference, wherein difference occurs corresponding to the formula according to table 4 (VII) in mode position 1,2,3,5,8,10,12,14,15,17 or 18.Difference can also include between the mode position of table 4 Room, insert or exchange.
The OgLuc misfolded proteins of the present invention or fusion protein can be by recombination methods or by solid state chemistry peptide symthesis method Prepare.Such method is as known in the art.
The method used and test kit
The compound of the present invention and albumen can use luciferase and luciferase substrate, such as, appointing of coelenterazine Where method uses.Such as, it can be used in Luminescence photography, the method uses the analog of coelenterazine to detect sample In one or more molecules, such as, enzyme, the cofactor of enzymatic reaction, zymolyte, enzyme inhibitor, enzyme activator or OH base, or One or more conditions, such as, Redox Condition.Sample can include animal (such as, vertebrates), plant, fungus, physiology Solution (such as, blood, blood plasma, urine, mucous secretion and similar physiological solution), cell, cell lysate, cell supernatant Or the cell fraction (such as, subcellular fraction) of purification.Can be to the existence of such molecule, amount, spectral distribution, Launch Dynamics Or specific activity carries out detecting or quantitatively.Can detect in the solution or quantitative molecular, described solution includes multi-phase solution (such as, breast Change liquid or suspension) or solid carrier (such as, granule, capillary tube or detection container).In some embodiments, OgLuc becomes Body can be used for measuring to detect enzyme interested, such as, CYP450 enzyme, MAO A enzyme or B enzyme, caspase based on luminous Deng.Novel coelenterazine can be used together with luminescent protein such as aequorin, Xi albumen or iPhotina.Implement at some In scheme, OgLuc variant can be used as the energy donor of another molecule (such as, fluorogen, chromophore or nanoparticle).
Present invention also offers the polynucleotide of encoding transcription report.In some embodiments, OgLuc variant or its Fragment can be operable with transcription regulating nucleotide sequence (such as, one or more enhancers, promoter, transcription terminator or a combination thereof) Ground connects to form expression cassette.Such as, OgLuc variant operationally can connect with minimal promoter and cAMP-response element (CRE) Connect.
The albumen of the present invention can be used as biosensor, such as, OgLuc variant, its another molecule (such as, one or Multiple molecules interested) in the presence of, or under certain conditions, there are one or more activity changed.With interested Interaction of molecules after or under certain conditions, biosensor experience conformational change or change chemically, cause luminescence The change of enzymatic activity, such as, specific activity, spectral distribution or Launch Dynamics.Such as, the OgLuc variant of the present invention, such as, The variant of circulation exchange, it may include with the region of interaction of molecules interested.Alternatively, such as, OgLuc variant can be with energy Amount receptor phase coupling, such as, fluorescin, and include the interaction changing the efficiency of the energy transfer from enzyme to energy acceptor Region.Such as, biosensor can be generated with detection by being inserted in OgLuc variant sequence thereof in suitable sensor region Protease, kinases, part, associated proteins such as antibody, cyclic nucleotide such as cAMP or cGMP, or metal such as calcium.Can be by one Plant or multiple sensor region is inserted into the C-end of peptide sequence, N-end and/or one or more suitable position, wherein Sensor region includes one or more aminoacid.In the case of the OgLuc variant of circulation exchange, sensor region can be inserted Enter between the N-end and C-end of parental generation OgLuc variant.Additionally, one or all of the sensor region inserted can be wrapped Include Linker amino acid with the remainder phase coupling by sensor with OgLuc variant.The example of luciferase biosensor exists U.S. Patent Application Publication No. 2005/0153310 and No. 2009/0305280 and PCT Publication the WO2007/120522nd Disclosed in A2, its each item is incorporated herein by reference.
In different embodiments, OgLuc variant disclosed herein can be used for shifting (such as, at biology energy During luminescence resonance energy transfer (BRET) is analyzed) to energy acceptor.Such as, OgLuc variant used in BRET analyzes can be used In determining whether two molecules can be combined with each other in cell or position altogether.Such as, OgLuc variant can be used as with interested The bioluminescence donor molecule of molecule or protein combination is to build the first fusion protein.In different embodiments, first melts Hop protein comprises OgLuc variant and albumen interested.In different embodiments, comprise the first fusion of OgLuc variant Albumen can be used for BRET and analyzes in the system including but not limited to cell lysate, complete cell and living animal with detection Albumen/protein-interacting.In different embodiments,Can be used as fluorescence acceptor molecules.Real at some Execute in scheme,Can be with the second albumen interested or OgLuc variant fusion.Such as OgLuc variant can be withMerge, express in cell or animal, and utilize fluorescencePart is such asTMR part is marked.Fusant can be excited to fluoresce in the presence of Premeabilisation of cells OgLuc substrate subsequently. In some embodiments, utilize the OgLuc variant combined with fluorescin can carry out BRET, described fluorescin include but It is not limited to green fluorescent protein (GFP) or red fluorescent protein (RFP) or fluorescent marker includes that fluorescein, rhodamine be green, Russia Strangle that ridge is green or Alexa488, only several non-limiting examples.
In different embodiments, the OgLuc variant of the present invention and novel coelenterazine can be used for protein complementation and measure (PCA) to detect the interaction of two biomolecule such as polypeptide.Such as, the OgLuc variant of the present invention can allow separation Site be divided into two fragments, and each fragment of the OgLuc variant separated can be interested be considered to interact One of a pair polypeptide (such as, FKBP and FRB) is merged.If two peptide species interested interact the most really, OgLuc Fragment then enters into close to each other to reconstitute functional, active OgLuc variant.In some embodiments, utilize The novel coelenterazine of natural or known coelenterazine or the present invention can detect and measure the activity of reconstruct OgLuc variant subsequently. In some embodiments, the OgLuc variant of division can be used for and lac-Z (Langley etc., PNAS72:1254-1257 ) or ribonuclease S (Levit and Berger, J.Biol.Chem.251:1333-1339 (1976)) is similar (1975) More general complementary system.In some embodiments, can be by known complementary with another OgLuc Variants Fragments (naming as " B ") OgLuc Variants Fragments (naming as " A ") and target protein merge, and can be via in the cell or the cell lysate that comprise fragment B The fusant of luminous monitoring gained.In some embodiments, the source of fragment B can be identical cell (such as, if On another plasmid gene of fragment B being inserted in the genome of cell or be contained in cell) or its can be derived from The lysate of another cell or the albumen of purification.In some embodiments, may utilize fragment B and solid carrier can be attached to Polypeptide such asBetween fusion catch or fix this identical fusion protein (fragment A).Real at some Executing in scheme, luminescence can be used for proving the amount of the material successfully catching or quantitatively being caught.
In different embodiments, OgLuc variant and/or the novel coelenterazine of the present invention can be used for quantitative coelenterazine. In some embodiments, coelenterazine (such as, natural or known coelenterazine, or the novel coelenterazine of the present invention) can be used as The probe of specific chemical-biological activities such as apoptosis and drug metabolism.In some embodiments, by interested specific Enzyme " pre-coelenterazine " or " front substrate " that it can be worked can be by coelenterazine concentration and specific enzymatic activity phase coupling.? In some embodiments, pre-coelenterazine is directly can not to support luminescence when combining with luciferase but can be by spy interested Fixed enzyme is converted into the molecule of coelenterazine through catalysis processing.In some embodiments, can be by institute in such as drug metabolism Method for enzyme, such as, cytochrome P 450 enzymes, monoamine oxidase, MAO and glutathione s-transferase；And apoptosis, such as half Guang aspartase.Such as, coelenterazine (such as, natural or known coelenterazine, or the novel coelenterazine of the present invention) can be modified To comprise the group of cleavable, such as 6 '-O-methyl.In some embodiments, when with specific cytochrome P 450 enzymes one Rising when hatching, 6 ' O-methyl are cleaved, and pre-coelenterazine is converted into the coelenteron of OgLuc variant detection of the available present invention Element.In some embodiments, pre-coelenterazine can with support needed for luminescence other components (such as, luminescent protein such as this Bright OgLuc variant) it is combined to provide single reagent and homogeneous determination.Such as, when adding reagent in sample, with front Coelenterazine is converted into coelenterazine and generates luminescence.In different embodiments, for can with from pre-coelenterazine generation chamber intestinal Other enzymes that element is associated, little molecule or other cell processes can develop similar mensuration.
In some embodiments, OgLuc variant and/or the novel coelenterazine of the present invention can be used as genetic transcription report System.In some embodiments, OgLuc variant can be from luciferase (such as, the red Pleonomus of the light launching different wavelength Luciferase (CHROMA-LUCTM；Promega Corp.)) MULTIPLE COMPOSITE.Such as, if the OgLuc variant of the present invention is used as function Report, then red CHROMA-LUCTMLuciferase can be used for controlling the nonspecific action to genetic regulation or for turning Dye efficiency is standardized.In some embodiments, the photometer with wavelength differentiation optical filter is utilized easily to resolve From OgLuc variant (about 460nm) and red CHROMA-LUCTMThe luminescence that (about 610nm) produces, allows to measure from identical sample Two kinds of signals of product.In another example, the OgLuc variant of the present invention can be used as transcribed reporter and with measure institute in reagent The luciferase launching different wave length contained matches.Such as, the OgLuc variant of the present invention can be used as transcribed reporter and water Female luminescent protein or the LUC Photinus pyralis LUC Photinus pyralis FL biosensor pairing of cAMP circulation exchange, or match with the two, with inspection simultaneously Survey the multiple approach in simple sample.In this system, such as, aequorin can be used for detecting and/or measuring calcium, raw Thing sensor is used for detecting and/or measuring cAMP, and OgLuc variant is used for monitoring downstream gene expression.In another example, OgLuc variant can be other with one or more luciferase be used together, wherein can by use selective enzyme inhibitor And measure the luminescence of every kind of luciferase respectively.Such as, the luminescence of the first luciferase can be eased up adding suitable substrate Rush and measure after liquid, then adding suitable substrate and buffer subsequently and one or more have choosing for the first luciferase The second luciferase is measured after the inhibitor of selecting property.In another example, measure luciferase contained in reagent and can be used for Measuring the specific aspect of cytophysiology, such as, ATP is to assess cell survival in measurement, or measures Caspase Activity To assess apoptosis.
In different embodiments, the OgLuc variant of the present invention be difficult to transfectional cell series or may even regardless of With son of giving a report in the primary cell (such as, stem cell or HepG2 cell) split.Due to its high signal intensity, work as transfection efficiency Time low, the OgLuc variant of the present invention will enable luminescence and can detect.In some embodiments, OgLuc variant can be the most right The cell (such as, sensitive or to transfection reagent the interpolation of its DNA concentration to increasing is sensitive) of the condition responsive relevant to transfection Middle with son of giving a report.So, in different embodiments, due to the luminescence of enhancing of the OgLuc variant of the present invention, utilize relatively Sensitivity cell is had by time after low DNA concentration, less transfection reagent and/or the shorter transfection started before measuring and making There is the toxicity burden of reduction can realize the luminescence of enough levels.In different embodiments, sending out of the enhancing of OgLuc variant Light allows to detect signal at later time point.In other other embodiments, OgLuc variant can be used as singly copying sky So report of promoter.
In different embodiments, the OgLuc variant of the present invention can be used as the fusion tag of target protein interested, makees Approach for the intracellular level of monitoring target protein.In some embodiments, OgLuc variant should in can be used for monitoring participation cell Swash the specific albumen of response pathway (such as DNA damage, oxidative stress, inflammation), as detecting different types of stimulus object The approach that may act on played in these approach.In some embodiments, OgLuc variant also acts as monitoring target protein The instrument that cell is transported goods for sale.Such as, OgLuc variant also can merge with viral genome (such as, HIV, HCV) thus can be with effectively Antiviral agent process after monitor the titre levels in cell, i.e. appeal.In some embodiments, variant also can be with green Fluorescin (GFP) orMerge (in addition to target protein) for fluorescence-activation cell sorting (FACS) with Identify high-expression clone.
In different embodiments, the signal of the enhancing of OgLuc variant and the small size of OgLuc gene can be conducive to table Reach the qualification of firm, the stable cell line of the OgLuc variant of the present invention of Cytoplasm form or secreted form.Relatively small Gene order can reduce the probability being incorporated into the genic instability caused in cellular genome by foreign DNA.
In different embodiments, the OgLuc variant of the present invention can be integrated into multiple different immunoassay concept In.Such as, OgLuc variant can fusion anti-with first or two to provide for the detection method of specific analyte.As another Individual example, OgLuc variant can merge with protein A or Protein G, and then fusant can be used for what detection was combined with specific analyte Specific antibody.As another example, OgLuc variant can be combined and be used for detection and specific analyte with streptavidin In conjunction with specific biotinylated antibody.As the example that another is other, the complementary fragment of OgLuc variant can anti-with one and Two anti-fusions, a wherein said specific analyte of anti-identification, and described two anti-identifications one resist.In some embodiments, OgLuc Variant Activity will reconstitute in the presence of analyte.As the example that another is other, OgLuc variant can be with analysis Thing (such as, prostaglandin) is conjugated and is used for competitive sandwich ELISA form.The OgLuc variant conjugated with analyte also may be used For detection can the antibody of bound analyte, wherein combine activity and allow OgLuc variant to be optionally connected with antibody.Utilize It is luciferase immunity that sea pansy belongs to luciferase for measuring the example of the patient antibodies's titre for the former target of antibody quantitatively Co-precipitation system (Burbelo etc., Expert Review ofVaccines9 (6): 567-578 (2010)).
In different embodiments, the OgLuc variant of the present invention and novel substrate can be used for detecting sending out in living cells Light.In some embodiments, OgLuc variant can express in cell (as report or other), and by coelenterazine (example As, novel coelenterazine, such as PBI-3939) process cell, it, by the cell in infiltration cultivation, reacts with OgLuc variant and gives birth to Become luminescence.In addition to as Premeabilisation of cells material, PBI-3939 shows the life suitable with natural coelenterazine about cell survival The thing compatibility.In some embodiments, can synthesize the natural coelenterazine in known raising medium stability comprise chemistry Modify PBI-3939 form and use it for report based on OgLuc variant more firm, living cells measure.Again In some other embodiments, utilize different microscopys and imaging technique can measure the OgLuc variant that comprises the present invention and/ Or the sample (including cell, tissue, animal etc.) of novel coelenterazine.In other other embodiments, secreting type OgLuc Variant is expressed in cell as the part of living cells report subsystem.
In different embodiments, OgLuc variant disclosed herein and/or novel coelenterazine can be as test kits Part provides.Test kit can include a kind of or different OgLuc variant (with polypeptide, polynucleotide or Both forms) and/or coelenterazine, and suitable reagent and user can be made to carry out all those measure as disclosed herein Description.Coelenterazine can be any one of natural, known or novel coelenterazine disclosed herein.Test kit May also include one or more buffer, than as disclosed herein those.
The luciferase of coding modification and the carrier of its fusant and host cell
Once be prepared for encoding OgLuc variant or its fragment (such as have luminescence activity fragment or can be with another molecule Complementary to cause the fragment of luminescence activity, or it has the fusant of luminescence activity) required nucleic acid molecules, volume can be prepared Code OgLuc variant or its fragment (such as, for complementary fragment or its there is the fusant of luminescence activity) expression cassette.Example As, will include that (such as, one or more strengthen for the nucleic acid molecules encoding the nucleotide sequence of OgLuc variant and transcription regulating nucleotide sequence Son, promoter, transcription terminator or a combination thereof) it is operably connected to form expression cassette.Can be by nucleic acid molecules or expression cassette Importing in carrier, such as, plasmid or viral vector, it optionally includes selectable marker gene, and is introduced in sense The carrier of the cell of interest, such as, prokaryotic cell such as escherichia coli, streptomyces certain (Streptomycesspp.), bud Spore Bacillus certain (Bacillus spp.), staphylococcus certain (Staphylococcusspp.) are thin with similar protokaryon Born of the same parents, and eukaryotic cell, including plant (dicotyledon or monocotyledon), fungus (include yeast, such as, Pichia sp. Belong to (Pichia), saccharomyces cerevisiae belongs to (Saccharomyces) or Schizosaccharomyces (Schizosaccharomyces)) or suckling Zooblast, its lysate or can import in vitro transcribe/translate in mixture.Mammalian cell includes but not limited to cattle Section animal, goat, caprid, Canis animals, felid, non-human primate cell, such as simian cells and the mankind Cell.It is thin that mammal cell line includes, but not limited to CHO, COS, HEK293, HeLa, CV-1, SH-SY5Y and NIH3T3 Born of the same parents, although it be also possible to use other cell lines substantial amounts of.
The expression of coded OgLuc variant can be by any promoter can expressed in prokaryotic cell or eukaryotic cell Promoter including synthesis is controlled.Prokaryotic promoter includes, but not limited to SP6, T7, T5, tac, bla, trp, gal, lac Or maltose promoter, including any fragment with promoter activity.Eukaryotic promoter includes, but not limited to composing type and opens Mover, such as, viral promotors such as CMV, SV40 and RSV promoter, and regulatable promoter, such as, inducible Or repressible promoters such as tet promoter, hsp70 promoter and the promoter of synthesis regulated and controled by CRE, open including having Any fragment of promoter activity.The expression of coded OgLuc variant also can be controlled by transcribing rear program, is such as added by RNA The program that the regulation and control of work or translation are regulated and controled, such as, drops by RNAi, miRNA, shRNA, siRNA or by RNA or albumen Solve.The nucleic acid molecules of the present invention, expression cassette and/or carrier can be introduced in cell by any method, include but not limited to, Conversion, electroporation, microinjection, lipofection and the similar method of calcium mediation.
The sequence of optimization of coding OgLuc variant and carrier and host cell
Also provide for is to include OgLuc variant, its function fragment or the nucleotide sequence of its fusion protein that code book is invented The nucleic acid molecules (polynucleotide) of separation.In some embodiments, the nucleic acid molecules of separation includes at least one Selected host expresses the nucleotide sequence being optimized.The sequence optimized includes codon optimized sequence, i.e. in one The codon more frequently used relative to another kind of organism (such as, outbreeding organism body) in organism, and modify to add Add or modify Kozak sequence and/or intron, and/or to remove unwanted sequence, such as, potential transcription factor combines Site.When being directed in host cell, the sequence of such optimization can provide the expression of enhancing, such as, the level increased Protein expression.The example of sequence optimized is disclosed in U.S. Patent No. 7,728,118 and U.S. Patent Application Publication the No. 2008/0070299, No. 2008/0090291 and No. 2006/0068395, its each item is incorporated herein by.
In some embodiments, polynucleotide include the nucleotide sequence of the OgLuc variant that code book invents, this nucleic acid sequence Row are optimized for the expression in mammalian host cell.In some embodiments, the polynucleotide of optimization are not Again with corresponding unoptimizable sequence hybridization, such as, under moderate or high stringency not with unoptimizable sequence hybridization.Art Language " strictly " is for the condition of the existence of temperature, ionic strength and other compounds of fingering row nucleic acid hybridization." the tightest Lattice " under the conditions of, nucleic acid base pairing will occur over just between the nucleic acid fragment with high-frequency complementary base sequence.So, When needs nucleic acid does not hybridizes or anneals together complete complementary mutually, often use the condition that " moderate " or " low " is strict.This Substantial amounts of equivalent condition can be used known to field to constitute moderate or Low stringency conditions.
In some embodiments, polynucleotide have at least 90% with corresponding unoptimizable sequence, such as, are less than The nucleic acid sequence identity of 80%, and optionally coding has at least 60% with the polypeptide coded by unoptimizable sequence, such as, The aminoacid sequence of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% Homogeneity.Additionally provide construct (the such as expression cassette of the nucleic acid molecules (such as, there is the nucleotide sequence of optimization) including separation And carrier), and include the test kit of the nucleic acid molecules, construct or the carrier that separate.
By include the nucleic acid molecules of the nucleotide sequence of OgLuc variant, its fragment or its fusant that code book is invented for Expression in specific host cell is optionally optimized, and the most optionally the most one or more with transcription regulating nucleotide sequence Enhancer, promoter, transcription terminator or a combination thereof are operably connected to form expression cassette.
In some embodiments, the nucleotide sequence of OgLuc variant, its fragment or its fusant of code book invention passes through Substitute codon to be optimized, such as, be used in specific (selected) cell the codon preferentially used to substitute parent For the codon of at least 25% in OgLuc sequence.Preferably codon has relatively high codon in selected cell Use frequency, and the relatively little of of transcription factor in the presence of preferably it result in selected host cell is transcribed Factor binding site, and other unwanted structure attributes relatively little of.The example of unwanted structure attribute includes, but does not limits In, restriction enzyme sites, eucaryon sequential element, vertebrate promoter assembly and Binding site for transcription factor, response element, large intestine Bacillus sequential element, mRNA secondary structure.So, the codon usage frequency that the nucleic acid product of optimization can have by increasing is led The expression of the level increased caused, and transcribed by unsuitable caused by the unwanted transcription regulating nucleotide sequence of the number reduced The risk of the reduction of behavior.
Separate and optimize nucleic acid molecules 30%, 35%, more than 40% or more than 45%, such as 50%, 55%, The codon composition different from corresponding wild type nucleic acid sequence can be had on 60% or more codon.Used by the present invention The codon of example be in specific organism for the identical aminoacid codon more other than at least one frequently Those codons that ground uses, and, in some embodiments, it is not the low utilization rate codon in this organism and the most not It it is the low utilization rate codon in the clone of the expression for nucleic acid molecules or the organism of screening.Further, for some amino The codon of acid (that is, having those aminoacid of three or more codons) can include than other (not preferred) codons The two or more codons more frequently used.In a kind of organism, ratio more frequently uses in another kind of organism Codon existence in nucleic acid molecules result in when being imported to by the nucleic acid molecules using those more frequently used codons Time in the cell of organism, with the expression of the nucleotide sequence higher than the wild type in those cells or parental generation in those cells Level is expressed.
In some embodiments of the present invention, different codons is that more frequently used those are close in mammal Numeral, and in other other embodiments, different codons is those codons more frequently used in plant. It is as known in the art for the different preferred codons of organism, for example, with reference to http: // www.kazusa.or.jp./codon/.Certain types of mammal, such as people, can have one and another type of suckling The a set of preferred codon that animal is different.Similarly, certain types of plant can have a set of with another type of plant not Same preferred codon.In one embodiment of the invention, the major part of different codons is the host wanted Preferred codon in cell.It is ability for including the preferred codon of organism of mammal (such as, people) and plant Known (such as, Wada etc., Nucl.Acids Res., 18:2367 (1990) in territory；Murray etc., Nucl.AcidsRes., 17:477 (1989)).
Embodiment
The synthesis of reference example 1 α amino nitrile (compound 1):
Flask is filled sodium sulfite (71.4mmol) and 17mL water.The 14mL oxolane of aldehyde (69.3mmol) will be contained (THF) solution is to keep internal temperature speed below 60 DEG C to be added drop-wise in above-mentioned flask.At room temperature by the suspension of gained Liquid stirs 40 minutes, and adds Ammonia (4.85mL) the time of 2 minutes.The solution of gained is carried out magnetic force stir Mix, heat 1 hour at 60 DEG C in oil bath simultaneously, and the most at room temperature stand overnight.By solution in ice salt water-bath cold But until measured internal temperature is below 5 DEG C.By the 14mL aqueous solution of sodium cyanide-containing (71.4mmol) 30 minutes time Between add in above-mentioned solution.The mixture of gained is stirred 20 minutes at about 10 DEG C, stirs 2 hours at 30 DEG C, and Stir 18 hours under room temperature.Reactant mixture is extracted in the ether of three parts of 200mL, and in anhydrous sodium sulfate, is dried merging Extract.Mixture is filtered and solution is cooled down 20 minutes in ice bath, hydrogen chloride gas is added to the molten of this stirring Until stopping precipitation in liquid, and suspension is stirred 1 hour.It is isolated by filtration solid the ether rinse with three parts of 50mL. It is dried in a vacuum material, it is thus achieved that the white solid (69%) of 6.4g (47.5mmol).Modification of program from: Freifelder and Hasbrouck, " Synthesis of Primary1,2-Diaminesby Hydrogenation of alpha- Aminonitriles, " Journal of the AmericanChemical Society, 82 (3): 696-698 (1960).
The synthesis of reference example 2 2-oxo-2-hyacinthin oxime (compound 2)
Flask is filled potassium tert-butoxide (58mmol) and the 63mL tert-butyl alcohol.Stirring mixture is until forming solution, and dropping contains The 35mL t-butanol solution of suitable benzophenone (50mmol) was time of 15 minutes.Reactant mixture is stirred 1 hour, and Pure amyl nitrite (75mmol) is added the times of 5 minutes.The heptan completing and utilizing 100mL of monitoring reactant mixture Alkane is diluted.By the solid (38mmol) of collected by suction gained and be dried under vacuum until constant weight.Modification of program from: Hagedom etc., Chem.Ber., 98:193 (1965).
The synthesis of reference example 3 pyrazines derivatives (compound 3)
By 3-mouth flask fitting temperature meter, barrier film and argon pipeline.By amino nitrile (47.5mmol), dry pyridine (190mL) add to wherein with oxime (61.75mmol).Thoroughly stirring mixture 15 minutes, and divide 5 parts to add the time of 35 minutes Add tetrachloro (double-pyridine) titanium complex (94.9mmol), it is ensured that internal temperature is maintained at less than 40 DEG C.After having added, in room Under temperature, reactant mixture is stirred overnight.Reactant mixture is added slowly to fraction the solution (174mL of sodium bicarbonate Water contains 21.75g) in.The mixture of gained thoroughly stirred 15 minutes and add 80g kieselguhr.Suspension is stirred 30 points Clock is also filtered by Buchner funnel.Filtrate is moved on to separatory funnel, and filter cake is suspended in the methanol of 400mL.Will mixing Thing stirs 30 minutes and again filters.Repeat this process totally four times.Methanol filtrate is combined and concentrates, residue is dissolved in In the ethyl acetate (EtOAc) of 200mL.Solution is added in the separatory funnel containing original filtrate, and with three parts of 100mL's EtOAc extracts mixture further.The extraction merged with the saturated sodium carbonate of two parts of 100mL and the brine of two parts of 100mL Take thing.Evaporation of organic solvent, it is thus achieved that brown oil slightly aoxidize pyrazine.Material is dissolved in the methanol of 3mL, adds the dichloro of 89mL Methane (DCM).In this solution, add zinc powder (80.7mmol), and mixture is cooled down until internal temperature reaches 15 in ice bath ℃.Process mixture with glacial acetic acid (3mL) and in oil bath, heat the internal temperature of 40 minutes to 30 DEG C.Reactant mixture is cold But filter to room temperature and by diatomaceous bedding and padding.Rinse filter cake with DCM, and wash with saturated sodium bicarbonate aqueous solution The filtrate merged.By utilizing heptane/EtOAc gradient by chromatograph, crude product to be purified on silica gel.This obtains brown 2.9g (29%) pyrazine of solid.Modification of program is from Kishi etc., " The structure confirmation of Thelight-emitting moiety of bioluminescent jellyfish. " Tetrahedron Lett., 13 (27): 2747 (1972).
The synthesis of reference example 4 coelenterazine
The following compound of method A:(can be synthesized by method A: compound PBI-3840, PBI-3886, PBI-3857, PBI-3887, PBI-3913, PBI-3894, PBI-3896, PBI-3897, PBI-3841 and PBI-3842)
Flask is filled pyrazine (8.25mmol), acetone acid (14.0mmol), camphorsulfonic acid (0.8mmol) and anhydrous 2-first Base THF (150mL).Condenser is installed on this flask and there is the Soxhlet extractor of 4 angstroms of molecular sieves, and reactant mixture is existed The oil bath of 110 DEG C is heated 18 hours.By molecular sieve with fresh replacement, and persistently backflow 24 hours.By reactant mixture mistake Filter and concentrate, residue is dissolved in EtOAc (200mL).With the saturated sodium bicarbonate solution of three parts of 25mL, the 0.1M of 100mL Sodium-acetate buffer, pH5 and 100mL brine solution.In magnesium sulfate, it is dried solution, filters and concentrate to obtain Thick enamine/the acid of 2.3g (6.2mmol, 75%).This material is dissolved in anhydrous THF (30mL), and by solution in ice water bath Cool down 10 minutes.It is added to carbodiimides (9.0mmol) and pure diisopropylethylamine (14.9mmol).After 10 minutes Remove psychrolusia, and reactant mixture is stirred at room temperature 3 hours.The vinegar of the 0.1M of 50mL is added in this reactant mixture Acid sodium buffer, pH5, and thoroughly stirring mixture 10 minutes.Extract this two-phase mixture with the EtOAc of three parts of 100mL, and use The extract that brine merges.Concentrate organic solvent, and by utilizing DCM/ methanol gradient to be come by chromatograph on silica gel Purification residue.This obtains the dehydration coelenterazine of 336mg (0.94mmol, 16%) red solid.This material is suspended in 10mL This mixture is cooled down in methanol and in ice bath.The time of 1 hour wherein with three parts add sodium borohydrides (100mg, 2.6mmol).It is stirred for reactant mixture 30 minutes, and drips pure glacial acetic acid until reaching pH5.Concentrate solution and use 15mL Water grinding residues.By sucking filtration separation solid and be dried under vacuum some hours solid to obtain 318mg (94%) yellow The thick coelenterazine of body.Modification of program is from Kakoi and Inoue, Chem.Lett.11 (3): 299-300 (1980).
The following compound of method B:(can be synthesized by method B: compound PBI-3882, PBI-3932, PBI-3881)
Flask is filled Biformyl (2.2mmol), Aminopyrazine (1.1mmol), ethanol (20mL), 12N hydrochloric acid (0.6mL) With water (1mL).Reactant mixture heated under reflux 24 hours and concentrate.By residue by utilizing DCM/ methanol gradient, Silica gel is purified by column chromatography.The coelenterazine of this black solid obtaining 100mg (0.25mmol, 23%) is produced Thing.Modification of program is from " the Squid bioluminescence.II.Isolation fromWatasenia such as Inoue Scintillans and synthesis of2-(p-hydroxybenzyl)-6-(p-hydroxyphenyl)-3,7- Dihydroimidazo [1,2-a] pyrazin-3-one " Chem.Lett., 4 (2): 141-4 (1975).
Method C: the synthesis of novel coelenterazine (following compound can be synthesized by method C: PBI-3939, PBI-3945, PBI-3889、PBI-4002)
Compound 4-(5-amino-6-benzyl pyrazine-2-base) phenol (Kishi can be prepared according to previously described method Deng., Tetrahedron Lett., 13:2747 (1972)；Mosrin etc., OrganicLetters, 11:3406 (2009)； Kakoi, Chem.Pharm.Bull., 50:301 (2002)).
The synthesis of 2-amino-3-benzyl-5-phenyl pyrazines.Round-bottomed flask is filled the 2-isonitroso of 5g (33.5mmol) 1-Phenylethanone., the 2-amino-3-hydrochloric acid phenylpropanenitrile of 6.7g (36.8mmol) and the anhydrous pyridine of 100mL.Mixture is cooled to- 20 DEG C and drip the TiCl of 4.6mL (40.0mmol)4.Reactant is kept 30 minutes at-20 DEG C, and is heated to 80 DEG C persistently 2.5 hour.Evaporation solvent, and residue is dissolved in 1L DCM.With saturated NaHCO3This solution is washed with saline.Evaporation is all Volatile matter, and residue is re-dissolved in ethanol (400mL).Add Raney Ni (2.0g, aqueous suspension), and big at 1 The stirring under hydrogen reactant of air pressure 5 days.By mixture by kieselguhr, and remove volatile matter.Residue is entered by silica gel Row chromatograph (heptane/DCM) is to obtain the 2-amino-3-benzyl-5-phenyl pyrazines of 2.5g (29%).
The synthesis of 2-amino-3-phenylpropanenitrile hydrochlorate.Round-bottomed flask is filled the sodium sulfite of 65g (0.624mol) Water with 150mL.The THF of the 150mL of the dropping hyacinthin containing 75g (0.624mol).After stirring 20 minutes, disposably add The 14M ammonium hydroxide of 37mL, and heat the mixture to 60 DEG C lasting 60 minutes.After being cooled to 0 DEG C, dilute with the water of 150mL Mixture, and drip the 100mL water of sodium cyanide-containing (27.5g, 0.560mol), keep internal temperature below 10 DEG C.After interpolation, Heat the mixture to 30 DEG C, continue 2 hours and utilize ether to extract.Dried with sodium sulfate, evaporate all volatile matters, and Residue is dissolved in the ether of 3.5L and with the ethanol HCl process of the 3.3M of 400mL.Filter the precipitate of gained and in vacuum In be dried with the product obtaining 55g (60%).
The synthesis of 3-(furan-2-base)-Acetylformic acid.3-(furan-2-base)-2-oxygen is added in the flask of 100mL For the 6N NaOH that propionate (940mg) and 23mL are cold.Insoluble mixture 5 minutes is stirred until dissolving in 90 DEG C of baths.Add Add cold 1N HCl until solution is acid (about 120mL).Solution is extracted with 2 × 50mL EtOAc.Wash with 40mL saline solution Wash the organic layer of merging, and use Na2SO4It is dried.Evaporation solution is to obtain 540mg brown solid.Pass through reversed-phase high-performance liquid chromatography (HPLC) gradient from 97% trifluoroacetic acid (TFA) be further purified solid to acetonitrile (ACN).
The synthesis of 3-(furan-2-base)-ethyl 2-oxopropanoate.To containing isomer (E/Z)-2-formamido-3-(furan Mutter-2-base) the 500mL flask of ethyl acrylate (5.0g) adds the 220mL 50/50 ethanol/water containing 1.4M (5%) HCl Freezing solution.After 5 hours, reaction separates between 200mLEtOAc and 30mL saline.By 2 × 50mL EtOAc extraction water Layer.The organic layer merged is washed with 1 × 50mL water and 1 × 50mL saline, and at Na2SO4In be dried.By organic layer and 26g diatom Soil coevaporation and on 80g silicon dioxide gold gradient from heptane to EtOAc, carry out eluting.Evaporate the component of suitable merging and obtain To 2.1g.
(E/Z) synthesis of-2-carboxyethyl-3-(furan-2-base) ethyl acrylate. in 500mL flask, add 50mL Ether, Cu2O (320mg) and Furan Aldehydes (5.2mL).Flask is cooled down in ice bath and adds 2-isocyano group ethyl acetate (5.3mL).After 1.5 hours, in reaction, add (5g) potassium tert-butoxide.After 4 hours, filter heterogeneous body reactant.Add The citric acid of 60mL30% and 20mL EtOAc also stir 10 minutes.Use 50mLEtOAc aqueous layer extracted.Do on anhydrous sodium sulfate The organic layer of dry merging.By EtOAc layer and 24g kieselguhr coevaporation and on 80g silicon dioxide gold gradient from heptane to EtOAc Eluting.Employ the slurry of yellow and do not carry out other purification.
2-oxo-3-(thiophene-2-base) propanoic acid. in the flask of 250mL, add (E/Z)-5-(thiophene-2-methylene) The cold 6N NaOH of imidazolidine-2,4--diketone (5.0g) and 100mL.Heat the mixture to 100 DEG C and continue 1 hour.Xiang Leng But solution adds the HCl of concentration until acidity (pH=1).With 8 × 50mL diethyl ether mixture.Wash with 50mL salt Wash the ether layer of merging, at Na2SO4Above it is dried and evaporates to obtain 3.36g solid.By utilizing α, α, α-benzotrifluoride is heavily tied Crystalline substance is further purified sample and obtains 1.63g.
(E/Z) synthesis of-5-(thiophene-2-methylene) imidazolidine-2,4-dione.Add in second in 250mL flask Uride (9.8g) and thiophene-2-formaldehyde (10g).Piperidines (9.6mL) is dripped in this mixture.Heat the mixture to 100 DEG C, Continue 1 hour and be subsequently poured in the 1N HCl of 300mL.Cross filter solid, wash with water and be dried in a vacuum and obtain 4.9g solid.
Step 1 adds suitable phenyl pyrazines-2-amine (100mg), suitable acetone in microwave vial (10mL) Acid (2 equivalent), DCM (1mL) and 1,1,1-trifluoroethanol (1mL), heated and stirred 30 minutes at 80 DEG C.By reactant at 2 grams On kieselguhr co-adsorption, and remove solvent in a vacuum.Kieselguhr is loaded on the spherical silica gel of 24g and with heptane to acetic acid The gradient elution of ethyl ester.Suitable fraction is merged and evaporates.
It is freezing in ice bath that step 2 will be dissolved in the step 1 of THF (0.5mL) material separated.Add acetic anhydride (25 μ L), dimethyl aminopyridine (8.5mg) and triethylamine (25 μ L).After 2 hours, remove the major part of THF in a vacuum.With Fructus Citri Limoniae aqueous acid (2mL) precipitated product of 30%.Wash solid with water (2mL) and be then dissolved in 3mL DCM.With 1 × 2mL water and 1 × 2mL brine DCM.Co-adsorption DCM layer on 2 grams of kieselguhr, and remove solvent in a vacuum.By silicon Diatomaceous earth is loaded on 12g spherical silica gel and with the gradient elution of heptane to DCM.Merge suitable component and evaporate.
Step 3 freezing in ice bath is dissolved in the material from step 2 of DCM (1mL).Methanol is added in this solution (0.5mL) the diethylene glycol dimethyl ethereal solution (325 μ L, 0.5M) with containing sodium borohydride.After 2 hours, add acetic acid (10 μ L), and should Solution promptly separates between the aqueous solution and DCM (2mL) of 30% citric acid (1mL).Co-adsorption DCM on 1 gram of kieselguhr Layer, and remove solvent in a vacuum.Kieselguhr is loaded on 4g spherical silica gel and with the gradient elution of DCM to EtOAc.Merge Suitable fraction also evaporates.
Step 4 (only work as R "=OAc) material in step 3 is dissolved in THF (200 μ L) and freezing in ice bath. The THF solution of 1 equivalent 1.35M Feldalat KM is added in solution.After 30 minutes, reactant is at DCM (1mL) and 30% citric acid (1mL) between separately.Co-adsorption DCM layer on 0.5g kieselguhr, and remove solvent in a vacuum.Kieselguhr is loaded into 4g's On spherical silica gel and with the gradient elution of DMC to EtOAc.Merge suitable fraction and evaporate.
Compound below method D:(can be synthesized by method D: compound PBI-3899, PBI-3900, PBI- 3925, PBI-3933, PBI-3946) general, with the 2-keto acid of 2 equivalents under the atmosphere of hydrogen in the presence of palladium catalyst Condensation Aminopyrazine.Purification the a-amino acid produced about intramolecular condensation activation subsequently produce corresponding Imidazopyrazines Ketone.
The synthesis of embodiment 5 8-benzyl-6-(4-hydroxyphenyl)-2-propyl imidazole also [1,2-α] pyrazine-3 (7H)-one
2-((3-benzyl-5-(4-hydroxyphenyl) pyrazine-2-base) amino) valeric acid.By 4-(5-amino-6-benzyl pyrazine-2- Base) phenol (100mg, 0.36mmol) with containing 2-oxopentanoic acid (84mg, 0.72mmol) ethanol (20mL) mixing.Add Pd/C (containing the activated carbon of 10% palladium, 40mg), and reactant mixture is heated to 65 DEG C.Pass through N2Gas makes air discharge, and by hydrogen Ball is applied to reaction flask.Continuously stirred reactant 4 hours.After cooling down, filter, and pass through purification by flash chromatography The solution (eluting solvent: the heptane containing 50%EtOAc) of gained and obtain the product (70mg, 52%) of yellow powder.1H NMR (300MHz, CD2Cl2, δ): 8.31 (s, 1H), 7.82 (d, 2H, J=9.0Hz), 7.31 (m, 5H), 6.92 (d, 2H, J= 9.0Hz), 5.34 (s, 2H), 4.20 (m, 1H), 1.10 (m, 2H), 0.98 (m, 2H), 0.87 (t, 3H)；MS(ESI)m/z378.3 (M+1)。
8-benzyl-6-(4-hydroxyphenyl)-2-propyl imidazole also [1,2-a] pyrazine-3 (7H)-one.By 2-((3-benzyl-5- (4-hydroxyphenyl) pyrazine-2-base) amino) valeric acid (49mg, 0.13mmol) is dissolved in DCM (10mL).Successively add pyridine (0.5mL) and N, N '-dicyclohexylcarbodiimide (54mg, 0.26mmol).It is slowly stirred at a room temperature reactant mixture 1 little Time.Evaporation solvent, and by purification by flash chromatography residue (eluting solvent: EtOAc to DCM to the DCM containing 10% methanol) Obtain the product (40mg, 86%) of yellow powder.1H NMR (300MHz, CD3OD, δ): 7.35 (m, 8H), 6.88 (d, J= 9.0Hz, 2H), 4.40 (s, 2H), 2.81 (t, J=7.5Hz, 2H), 1.81 (m, 2H), 1.02 (t, J=7.5Hz, 3H)；MS (ESI)m/z359.0。
The synthesis of embodiment 6 8-benzyl-2-butyl-6-(4-hydroxyphenyl) imidazo [1,2-a] pyrazine-3 (7H)-one
2-((3-benzyl-5-(4-hydroxyphenyl) pyrazine-2-base) amino) caproic acid.By 4-(5-amino-6-benzyl pyrazine-2- Base) ethanol (20mL) mixing of phenol (200mg, 0.72mmol) and ketone group caproic acid sodium salt containing 2-(220mg, 1.44mmol).Add Add Pd/C (containing the activated carbon of 10% palladium, 100mg) and some acetic acid, and reactant mixture is heated to 65 DEG C.Pass through N2Gas Hydrogen balloon is also applied to reaction flask by body air-out.Continuously stirred reactant 4 hours.After cooling down, filter and pass through The solution (eluting solvent: the heptane containing 50%EtOAc) of purification by flash chromatography gained and obtain yellow powder product (130mg, 46%).MS (ESI): m/z392.2 (M+1).
8-benzyl-2-butyl-6-(4-hydroxyphenyl) imidazo [1,2-a] pyrazine-3 (7H)-one.By 2-((3-benzyl-5- (4-hydroxyphenyl) pyrazine-2-base) amino) caproic acid (130mg, 0.33mmol) is dissolved in DCM (10mL).Successively add pyridine (0.5mL) and N, N '-dicyclohexylcarbodiimide (137mg, 0.67mmol).Stirring reactant mixture 1 the most lentamente Hour.Evaporation solvent, and by purification by flash chromatography residue (eluting solvent: EtOAc to DCM to the DCM containing 10% methanol) And obtain the product (110mg, 89%) of yellow powder.1H NMR (300MHz, CD3OD, δ): 7.30 (m, 8H), 6.88 (d, 2H), 4.40 (s, 2H), 2.84 (t, 2H), 1.77 (m, 2H), 1.51 (m, 2H), 0.89 (m, 3H)；MS(ESI)m/z374.3(M+1).
Embodiment 7 8-benzyl-2-ethyl-6-phenylimidazole also [1,2-a] pyrazine-3 (7H)-one (PBI-3925) Synthesis
2-((3-benzyl-5-phenyl pyrazines-2-base) amino) butanoic acid.By 3-benzyl-5-phenyl pyrazines-2-amine (200mg, 0.77mmol) with ethanol (20mL) mixing containing 2-Oxobutyric acid (157mg, 1.54mmol).Add the Pd/C (work containing 10% palladium Property charcoal, 100mg), and reactant mixture is heated to 65 DEG C.Pass through N2Gas air-out, and hydrogen balloon is applied to reaction Flask.Continuously stirred reactant 4 hours.After cooling down, filter, and by the solution (eluting of purification by flash chromatography gained Solvent: the heptane containing 50%EtOAc) and obtain the product (90mg, 34%) of yellow powder.1H NMR (300MHz, CD2Cl2, δ): 7.72 (s, 1H), 7.32-7.48 (m, 10H), 4.46 (s, 2H), 4.20 (m, 2H), 2.25 (q, 2H), 0.99 (t, 3H)；MS (ESI): m/z348.3 (M+1).
2-((3-benzyl-5-phenyl pyrazines-2-base) amino) butanoic acid is dissolved in DCM (10mL).Successively add pyridine (0.5mL) and N, N '-dicyclohexylcarbodiimide (137mg, 0.67mmol).Stirring reactant mixture 1 the most lentamente Hour.Evaporation solvent, and by purification by flash chromatography residue (eluting solvent: EtOAc to DCM to the DCM containing 10% methanol) And obtain the product (110mg, 89%) of yellow powder.1H NMR (300MHz, CD3OD, δ): 7.26 (m, 3H), 6.84-7.07 (m, 8H), 4.03 (s, 2H), 2.47 (q, J=9.0Hz, 2H), 0.96 (t, J=9.0Hz, 3H)；MS (ESI): m/z330.2 (M+ 1)。
Embodiment 8 8-benzyl-6-phenyl-2-(3,3,3-trifluoro propyl) imidazo [1,2-a] pyrazine-3 (7H)-one Synthesis
5,5,5-tri-fluoro-2-oxopentanoic acids.By 4,4,4-trifluoroacetic acid ethyl ester (1g, 5.88mmol) and ethyl oxalates (3.87g, 26.5mmol) is dissolved in ethanol.Sodium ethylate (21% is contained in ethanol, 2.09g) is added in solution, and stirs Reactant mixture 0.5 hour.Solvent distillation, and by EtOAc/ water extracted residues.Collected organic layer is also dried in sodium sulfate. After filtration, remove solvent and obtain the liquid of clarification.MS (ESI): m/z269.1 (M-1).Then liquid is dissolved in 3N HCl (20mL) in, and reaction mixture refluxed 4 hours.After cooling down, with EtOAc extractive reaction mixture.Collected organic layer And be dried in sodium sulfate.After filtration, remove solvent, and residue is directly used in next step.MS (ESI): m/ z169.7(M-1)。
5,5,5-tri-fluoro-2-((3-benzyl-5-phenyl pyrazines-2-base) amino) butanoic acid.By 3-benzyl-5-phenyl pyrazines- 2-amine (240mg, 0.92mmol) is with containing 5, and the ethanol (20mL) of 5,5-tri-fluoro-2-oxopentanoic acids (150mg, 0.88mmol) mixes Close.Add Pd/C (containing the activated carbon of 10% palladium, 100mg), and reactant mixture is heated to 65 DEG C.Pass through N2Gas discharges sky Gas, and hydrogen balloon is applied to reaction flask.Continuously stirred reactant 4 hours.After cooling down, filter, and by quickly The solution (eluting solvent: the heptane containing 50%EtOAc) of chromatogram purification gained and obtain yellow powder product (200mg, 54%).1H NMR (300MHz, CD2Cl2, δ): 11.45 (s, 1H), 10.20 (s, 1H), 7.94 (s, 1H), 7.34 (m, 10H), 5.34 (s, 2H), 3.96-4.23 (m, 2H), 3.02-3.28 (m, 2H)；FNMR:-76.3；MS (ESI): m/z416.1 (M+1).
Coelenterazine (R1=H, R2=-CH2CH2CF3).By 5,5,5-tri-fluoro-2-((3-benzyl-5-phenyl pyrazines-2-base) ammonia Base) butanoic acid (100mg, 0.24mmol) is dissolved in DCM (10mL).Successively add pyridine (0.5mL) and N, N '-dicyclohexyl carbon two Imines (100mg, 0.48mmol).Stirring reactant mixture 1 hour the most lentamente.Evaporation solvent, passes through flash chromatography Purification residue (eluting solvent: EtOAc to DCM to the DCM containing 10% methanol) and obtain yellow powder product (80mg, 87%).1H NMR (300MHz, CD2Cl2, δ): 7.36 (m, 11H), 3.43 (s, 2H), 1.60-1.92 (m, 4H)；FNMR:67.4 (t, J=18Hz)；MS (ESI): m/z398.2 (M+1).
Embodiment 9 8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazole also [1,2-a] pyrazine-3 (7H)-one (PBI-3939) synthesis
8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazole also [1,2-a] pyrazine-3 (7H)-one: utilize 3-(furan Mutter-2-base)-Acetylformic acid and 3-benzyl-5-phenyl pyrazines-2-amine synthesizes by method C as parent material.1H NMR (300MHz, dmso) δ 8.88 (s, 1H), 8.02 (d, J=7.9,2H), 7.61-7.38 (m, 6H), 7.37-7.14 (m, 3H), 6.38 (s, 1H), 6.26 (d, J=3.2,1H), 4.64 (s, 3H), 4.40 (s, 3H)；For C24H20N3O2 +The accurate matter calculated Amount m/z+382.16, surveys m/z+382.
Embodiment 10 8-benzyl-6-phenyl-2-(thiophene-2-ylmethyl) imidazo [1,2-a] pyrazine-3 (7H)-one (PBI-3889) synthesis
8-benzyl-6-phenyl-2-(thiophene-2-ylmethyl) imidazo [1,2-a] pyrazine-3 (7H)-one: utilize 2-oxo- 3-(thiophene-2-base) propanoic acid and 3-benzyl-5-phenyl pyrazines-2-amine are synthesized by method C as parent material.1H NMR (300MHz, dmso) δ 8.85 (s, 1H), 7.99 (d, J=6.8,2H), 7.63-7.02 (m, 10H), 6.94 (dd, J=3.5, 5.1,1H), 4.62 (s, 2H), 4.58 (s, 2H), 2.69 (impurity)；For C24H20N3OS+The exact mass m/z+ calculated 398.13, survey m/z+398.
Embodiment 11 8-cyclopropyl-2-(4-hydroxybenzyl)-6-phenylimidazole also [1,2-a] pyrazine-3 (7H)-one (PBI-3897) synthesis
8-cyclopropyl-2-(4-hydroxybenzyl)-6-phenylimidazole also [1,2-a] pyrazine-3 (7H)-one: utilize 3-ring third Base-5-phenyl pyrazines-2-amine and 3-(4-hydroxyphenyl)-Acetylformic acid synthesize as parent material Application way A.For C22H18N3O2 -The exact mass m/z-356.14 calculated, surveys m/z-356.
Embodiment 12 8-benzyl-2-methyl-6-phenylimidazole also [1,2-a] pyrazine-3 (7H)-one (PBI-3932) Synthesis
8-benzyl-2-methyl-6-phenylimidazole also [1,2-a] pyrazine-3 (7H)-one: utilize 1,1-dimethoxy propane- 2-ketone and 3-benzyl-5-phenyl pyrazines-2-amine synthesize as parent material Application way B.For C20H18N3O+Calculate Exact mass m/z+316.14, surveys m/z+316.
Embodiment 13 2-(4-hydroxybenzyl)-8-methyl-6-phenylimidazole also [1,2-a] pyrazine-3 (7H)-one (PBI-3896) synthesis
2-(4-hydroxybenzyl)-8-methyl-6-phenylimidazole also [1,2-a] pyrazine-3 (7H)-one: utilize 3-methyl-5- Phenyl pyrazines-2-amine and 3-(4-hydroxyphenyl)-Acetylformic acid synthesize as parent material Application way A.1H NMR (300MHz, dmso) δ 8.84 (s, 1H), 8.00 (d, J=7.6,2H), 7.47 (dd, J=8.6,16.2,3H), 7.17 (d, J= 7.3,2H), 6.69 (d, J=8.4,2H), 6.26 (s, 4H), 4.17 (s, 2H), 2.86 (s, 3H), 2.48 (s, 1H).
Embodiment 14 8-benzyl-2-(4-hydroxybenzyl)-6-phenylimidazole also [1,2-a] pyrazine-3 (7H)-one (PBI-3840) synthesis
8-benzyl-2-(4-hydroxybenzyl)-6-phenylimidazole also [1,2-a] pyrazine-3 (7H)-one: utilize 3-(4-oxybenzene Base)-Acetylformic acid and 3-benzyl-5-phenyl pyrazines-2-amine synthesizes as parent material Application way A.For C26H22N3O2 +The exact mass m/z+408.17 calculated, surveys m/z+408.
The synthesis of the shielded coelenterazine of embodiment 15 (stabilisation) (PBI-4377)
At room temperature in argon gas atmosphere to PBI-3939, containing potassium carbonate (1.1 equivalent) and the two of potassium iodide (1.1 equivalent) The mixture of methylformamide (DMF) adds the chloromethyl pivalate of 1 equivalent.Reaction process is monitored by thin layer chromatography, and After completing, ice bath cools down reactant mixture a few minutes, then adds the water of the volume identical with reaction volume.It is suitable to utilize The mixture of organic solvent (such as, EtOAc) extraction gained, and concentrate extract and obtain crude product.By on silica gel Carry out chromatograph and be further purified material.
Embodiment 16 8-benzyl-2-((1-methyl isophthalic acid H-imidazoles-2-base) methyl)-6-phenylimidazole also [1,2-a] Pyrazine-3 (7H)-one (PBI-4525), 8-benzyl-6-(4-hydroxyphenyl)-2-((1-methyl isophthalic acid H-imidazoles-2-base) methyl)-6- Phenylimidazole also [1,2-a] pyrazine-3 (7H)-one (PBI-4540) and 2-((1H-imidazoles-2-base) methyl)-8-benzyl-6-benzene The synthesis of base imidazo [1,2-a] pyrazine-3 (7H)-one (PBI-4541)
The anhydrous THF of 20mL is added under an argon atmosphere to the flask of the 2-methylimidazole derivant 1 or 2 containing 10mmol, And in dry ice/acetone batch, cool down solution to about-78 DEG C.In this cooling mixture, 9.3mmol is dripped in the time of a few minutes The solution of n-BuLi (2.46M is in hexane).At about-78 DEG C, stir the solution 30 minutes of gained, and added by syringe Add the compound 3 of 6.7mmol.Reactant mixture is stirred 3 hours and utilizes and add ammonium chloride solution saturated for 20mL and 20mL Saturated sodium bicarbonate solution carrys out cancellation.Remove cryostat, and after being heated up to room temperature, extract with the EtOAc of 3 × 100mL Mixture.It is dried (MgSO4) extract that merges, concentrate in a vacuum, and utilize silica gel (EtOAc/ heptane) to pass through column chromatography Purifying crude compound 4 or 5.
Microwave vial is filled the compound 8 or 9 of compound 6 and 7 and 2 equivalent of 100mg (1 equivalent).To this mixture The ethanol of middle interpolation 4.5mL and the HCl of the concentration of 0.25mL.In microwave at 100 DEG C reacting by heating mixture 1.5 hours. Gained reactant mixture is added in the EtOAc of 50mL and subsequently with saturated bicarbonate enzymatic solution and the salt of 20mL of 20mL Water washs.Concentration of organic layers in a vacuum, and utilize silica gel (ethanol/methylene) to be obtained by column chromatography purification residue Compound 10-12.
The stability of the novel coelenterazine of embodiment 17 and self-luminous characterize
Determine the stability of novel coelenterazine PBI-3939, PBI-3889, PBI-3945, PBI-4002 or PBI-3896 Characterize with self-luminous.Higher stability and relatively low self-luminous are the attracting technical characteristics in substrate/reagent.
For determining stability, by novel coelenterazine PBI-3939 of 20 μMs, PBI-3889, PBI-3945, PBI-4002 or PBI-3896, known coelenterazine-h or the known coelenterazine-hh of the natural coelenterazine of 30 μMs or 22 μMs are placed in containing 50mM CDTA, 150mMKCl, 50mM DTT, 35mM thiourea, 1%NP-9 (v/v) and 0.1% In the report reagent buffer of DF204.The different time span of the sample of duplication and so is hatched under room temperature (that is, 22-24 DEG C) After transfer to-70 DEG C.Collect and freezing all of sample after, melted and with without phenol red+0.1% 40 μ L DMEM in the 10 μ L bacteria cell cracking things containing OgLuc variant IV mix mutually.5 minutes reading samples after adding IV The luminescence of product.
“T90" represent at room temperature that is 22 DEG C, the time used of luminous signal decay 10% (that is, activity reduces 10%) Amount.The speed of luminous signal decay is determined from the slope of the linear fit of the data of ln RLU plotted over time (“T90"), it is calculated by below equation: t=ln (A/A0) ÷ (-k), wherein intensity during A=time t, A0During=time 0 Intensity, and k=decay speed.As shown in table 1, it is known that coelenterazine-h and-hh, novel coelenterazine PBI-3939, PBI- 3889, the T of PBI-3945, PBI-4002 and PBI-389690Value, higher than natural coelenterazine, shows that these coelenterazine are than natural chamber The compound that intestinal element is more stable.
For determining self-luminous feature, by HEK293 cell with 15,000 cell per well is at DMEM+10%FBS+ acetone acid Overnight incubation in salt.Remove culture medium and be used in add 10%FBS be independent of CO2Culture medium in dilution Fig. 2 shown in Every kind of novel coelenterazine, i.e. PBI-3939, PBI-3889, PBI-3945, PBI-4002, PBI-3841, PBI-of 20 μMs 3897, PBI-3896, PBI-3925, PBI-3894, PBI-3932 and PBI-3840, natural coelenterazine and known coelenterazine, Coelenterazine-h and coelenterazine-hh substitutes.Exist at once after adding substrateOn Luminometer (1 second/hole) Measure luminescence.Background luminescence is 154 ± 15RLU.Table 1 shows that self-luminous feature standardized for natural coelenterazine is (" spontaneous Light (about coelenterazine standardization) ").Coelenterazine-h has a more self-luminous than natural coelenterazine, but checked all its His coelenterazine has less self-luminous.
Table 1: utilize the stability experiment of the IV that different coelenterazine carries out and self-luminous to characterize
The toxicity of the novel coelenterazine of embodiment 18
The toxicity of novel coelenterazine is have studied in HEK293 cell.By HEK293 cell with 15,000/ hole at DMEM+ 10%FBS+ pyruvate grows overnight.Remove culture medium and be used in and be independent of CO containing 10%FBS2Culture medium in dilute Novel coelenterazine compound (or DMSO comparison) replace.After adding compound 24 hours, utilize according to the explanation of manufacturerMeasure reagent (Promega Corp.) and measure cell survival.And Luminometer measures luminescence on (1 second/hole).Table 2 show novel coelenterazine, known coelenterazine-h and coelenterazine-hh and Novel coelenterazine PBI-3939, PBI-3889, PBI-3841, PBI-3897, PBI-3945, PBI-4002 and PBI-3840 exist Toxicity in HEK293 cell.In addition to PBI-3840, novel coelenterazine has at least identical with coelenterazine-hh toxicity.Novel Some in coelenterazine have the toxicity identical with natural coelenterazine and coelenterazine-h.
Table 2: according toDifferent coelenterazine toxicity in HEK293 cell
Substrate Viability (about carrier (DMSO) reference standard) (%)
Natural coelenterazine 100
Coelenterazine h 100
Coelenterazine hh 87
PBI-3939 89
PBI-3889 90
PBI-3841 100
PBI-3897 100
PBI-3945 100
PBI-4002 100
PBI-3840 60
The Km of embodiment 19 PBI-3939
For determining the Km of PBI-3939, utilize according to the explanation of manufacturerProtein Purification System passes throughFusant purification OgLuc variant L27V is (described in embodiment 26 ) and without phenol red and 0.1%DMEM in dilute.50 μ L are measured buffer (100mM MES PH6,35mM thiourea, 0.5%NP-9 (v/v), 1mM CDTA, 2mM DTT and 150mM KCl) from different The PBI-3939 of amount adds in the enzyme that 50 μ L dilute (the final enzyme concentration of about 20pM), and when 3 minutes, measurement was sent out at 22 DEG C Light.As the data in Fig. 3 show, the Km of PBI-3939 is about 10 μMs.
The sign of embodiment 20 compound PBI-4525, PBI-4540 and PBI-4541
SCREENED COMPOUND PBI-4525, PBI-4540 and PBI-4541 ability to detect luminescence.In order to analyze, by 20 μMs Every kind of compound add to mensuration buffer (100mM MESpH6,35mM thiourea, 0.5%NP-9(v/ V), 1mM CDTA, 2mMDTT and 150mM KCl), it is adjusted to pH7 with 100mM HEPES pH7 and measures reagent to build. Then do not conform to phenol red and 0.1%DMEM in will measure the L27V02 enzyme of reagent and 36pM purification and (implement Described in example 25B) mixing.As comparison, use the mensuration buffer containing 20 μMs of PBI-3939 or PBI-4528.To Mensuration reagent adds in enzymatic mixture latter 3 minutes measures luminescence as described earlier.Table 3 indicates compound PBI- 4525, PBI-4540 and PBI-4541 can be used for detecting the luminescence from the luciferase utilizing coelenterazine.
Table 3
Compound RLU +/-
4525 20,655 1,006
4528 202,080 5,688
3939 9,808,880 307,565
4540 909 7
4541 5,676 80
Embodiment 21 OgLuc mode sequences
By having, the catalysis activity of common three dimensional structure and restriction is recognizable includes different luciferase kinds Enzyme family.Because enzyme family and other enzyme families have evolutionary history, they also will show the similarity of its three dimensional structure.Pass through The 26S Proteasome Structure and Function analysis of different modes, the present inventor has identified the OgLuc representative as decapods luciferase, have with The three dimensional structure that fatty acid binding protein (FABP) is the most similar, indicates the general character of evolutionary history.So, can be by decapods fluorescence Element enzyme is defined as having the characteristic three dimensional structure similar to FABP, and utilizes coelenterazine as substrate sending out with catalytic luminescence Penetrate.Other luciferases, such as, LUC Photinus pyralis LUC Photinus pyralis FL, sea pansy belongs to luciferase, bacterial luciferase and similar fluorescein Enzyme, has visibly different three dimensional structure, shows that it belongs to different enzyme families and does not have evolutionary history.Dinoflagellate (Dinoflagellate) luciferase has the three dimensional structure showing some similaritys with FABP, shows the evolution having History, but do not utilize coelenterazine as substrate, and thus it is not belonging to the enzyme family identical with decapods luciferase.
Because aminoacid sequence is conservative not as three dimensional structure, it is based only upon gene comparision and is probably tired to define enzyme family Difficult.Such as, even if FABP has distinctive barrel-shaped 3D shape, its aminoacid sequence more often show very low-level Sequence iden.While it is true, sequence iden may be used to indicate that the general character of three dimensional structure.If can be by the amino of two albumen Acid sequence is compared and is shown ＞ 30% sequence iden, preferably ＞ 40% sequence iden, and most preferably ＞ 50% Sequence iden, then three dimensional structure (Chothia and Lesk, EMBO J.5 (4): the 823-826 that two albumen will have like (1986)；Tramontano, Genomics, 4:402-405 (2003)).So, if based on its aminoacid sequence with OgLuc's The comparison of sequence, sequence iden ＞ 30%, preferably ＞ 40%, and most preferably ＞ 50%, and albumen may utilize coelenterazine As substrate with the transmitting of catalytic luminescence, then albumen is decapods luciferase.
Structure necessary to the characteristic three dimensional structure of maintenance enzyme family limits, the aminoacid sequence in enzyme family Some parts shows conservative (that is, higher sequence iden level) of higher amount.So, these conservative regions can be used Make the other evidence of common three dimensional structure total between two albumen.Conservative sequence pattern, also referred to as labelling, die body or Trace, can be generated by as known in the art artificial or computer method.Pattern is found in public database such as PROSITE(http://expasy.org/prosite；Sigrist etc., NucleicAcids Res.38 (supplementary issue 1): D161- D166(2010))。
Such as, conservative amino acid whose pattern can find when checking substantial amounts of known FABP.PROSITE (distribution 20.67, on October 5th, 2010) (accession number PS00214, is implemented in April nineteen ninety, and data are updated to comprise FABP pattern In April, 2006).This FABP pattern is crossed over 18 amino acid positions and is limited as follows:
[GSAIVK]-{FE}-[FYW]-x-[LIVMF]-x-x-{K}-x-[NHG]-[FY]-[DE]-x-[LIVMFY]- [LIVM]-N}-{G}-[LIVMAKR] (SEQ ID NO:329) (VI),
Wherein:
Employ amino acid whose standard IUPAC one-letter code.
Symbol " x " is used for accepting any amino acid whose position.
The optional aminoacid of site (such as, [ALT] generation is indicated by listing aminoacid between square brackets " [] " The probability of this position of table Ala, Leu or Thr).
By curly brackets " { } " indicate site go out specific amino acids existence (such as, AM} represent in position Any aminoacid in addition to Ala and Met).
Each sequence location (or the element in pattern) is adjacent and is separated by "-".
Each sequence location is referred to as " mode position ", and such as [GSAIVK] will be considered as the mode position of formula (VI) 1, FE} is considered as the mode position 2 of formula (VI), etc..
Although conservative sequence is caused due to common potential three dimensional structure, can allow for some of sequence pattern Do not destroy the change of three dimensional structure.Such as, for some members of FABP family, four site in PROSITE pattern It is found that difference.These other members of FABP family include 5 eggs met as false negative listed in PROSITE In vain, i.e. FABP pattern FABP not to be covered protein family member (UniProt database accession number FBP12_HUMAN, FABP1_FASGI, FABP2_FASHE, FABPL_SCHBI, RET5_BOVIN) and a known albumen with FABP folding (albumen database logs in numbering 2A02).Although OgLuc to FABP the most similar total three dimensional structure, natural and variant The sequence pattern of aminoacid sequence is the most different, has difference 5 positions with PROSITE pattern.In different enforcement In scheme, the pattern in OgLuc originates in the position of the 8th corresponding to SEQ ID NO:1.Amino acid replacement, lack or insert Mode entry sequence is calculated as a difference.
Combine the sequence information from these other FABP and OgLuc variants, can derive the sequence pattern of improvement:
[GSAIVK]-{FE}-[FYW]-x-[LIVMFSYQ]-x-x-{K}-x-[NHGK]-x-[DE]-x-[LIVMFY]- [LIVMWF]-x-{G}-[LIVMAKRG] (SEQ ID NO:330) (VII).
The sequence information used by this pattern of deriving is shown in table 4.1st row identify mode position (by N-end to C- End is listed；Modal length is 18 aminoacid), and the 6th row identify in OgLuc corresponding sequence location (according to SEQ ID NO:1 numbers).2nd row show PROSITE FABP pattern (formula (the VI)) element for each mode position.3rd row row Go out to be present in the aminoacid representated by non-PROSITE FABP pattern in FABP family member.4th lists and is present in In OgLuc (SEQ ID NO:1) or OgLuc variant not by the aminoacid representated by PROSITE pattern.5th list logical Cross pattern (" OgLuc pattern ") (formula of the improvement pattern information from the 2nd row, the 3rd row and the 4th row merged and build (VII)).7th lists the aminoacid in OgLuc corresponding to PROSITE FABP mode position.8th list corresponding to (GenBank logs in the aminoacid found in the dinoflagellate luciferase sequence (8 different species) of the position of the pattern of improvement Number 2021262A, AAA68491, AAC36472, AAV35379, AAV353800, AAL40676, AAL40677, AAV35378, AAV35377, AAV35381 and protein database accession number 1VPR).
The pattern (formula (VII)) of improvement is used as instruction (that is, the print of three-dimensional protein structure total between FABP and OgLuc Mark).However, it is not necessary to the strict consistent general character indicating three dimensional structure of this pattern.By examples given herein, i.e. Make pattern exists up to 5 changes, common three dimensional structure can be there is.Equally, such as, though dinoflagellate luciferase and FABP Have similar three dimensional structure with OgLuc, it has 4 differences with the pattern of improvement.
Such that make based on sequence similarity and utilize coelenteron the most luminous, albumen can be identified as decapods fluorescein Enzyme, it is possible to by there is the sequence pattern of improvement to its further identification simultaneously.Especially, if based on its aminoacid sequence with SEQ ID NO:1 or the comparison of its variant, sequence iden is ＞ 30%, preferably ＞ 40%, and most preferably ＞ 50%, and Albumen may utilize coelenterazine as substrate with the transmitting of catalytic luminescence, and originates in the position of corresponding to SEQ ID NO:1 the 8th The aminoacid sequence put is following:
[GSAIVK]-{FE}-[FYW]-x-[LIVMFSYQ]-x-x-{K}-x-[NHGK]-x-[DE]-x-[LIVMFY]- [LIVMWF]-x-{G}-[LIVMAKRG] (SEQ ID NO:330) (VII),
Have less than 5 differences, or no more than 4,3,2 or 1 difference, or more preferably indifference Different, wherein difference occurs in the mode position 1,2,3,5,8,10,12,14,15,17 or 18 corresponding to the formula (VII) according to table 4 Position in, then albumen is decapods luciferase.Difference may also comprise the room between the mode position of table 4 or insertion.
Table 4: protein sequence pattern
The generation of embodiment 22 OgLuc variant
Experimental detail
Except as otherwise noted, the other variant of the initial OgLuc variant sequence thereof with random permutation utilizes luring of fallibility The system GeneMorph II RandomMutagenesis Kit (Stratagene of the PCR-based become；Daughtery, PNAS USA, 97 (5): 2029 (2000)) according to explanation and saturated (Zheng etc., the Nucleic Acids in NNK site of manufacturer Research, 32:e115 (2004)) generate.
The other variant with specific sudden change of initial OgLuc variant utilizes direct mutagenesis based on oligonucleotide examination Agent box QuikChange Site-Directed MutagenesisKit (Stratagene；Kunkel, PNAS USA, 82 (2): 488 (1985)) generate according to the explanation of manufacturer.
The variant of gained is at the pF1K for expression based on T7 promoterCarrier (Promega Corp.) Environment in build.Alternatively, the variant of gained is at the pF4Ag carrier (form (Promega of the pF4A being obtained commercially Corp.) building in environment), it comprises modified and comprises with or without C-end(Promega Corp.；The T7 of escherichia coli ribosome binding site referred to herein as " HT7 ") and CMV promoter (Ohana etc., Protein Expression and Purification, 68:110-120 (2009)) to generate fusion protein.Such as, for obtaining C1+A4E Variant, carries out NNK saturation mutagenesis experiment in pF1K vector background.C1+A4E is generated in the pF4Ag vector background without HT7 Library.QC27, QC27-9a and IVY library is generated in the pF4Ag vector background with C-end HT7.Without HT7's PF4Ag vector background generates variant based on IV.Technology as known in the art is utilized to be used for converting KRX by the carrier of gained Escherichia coli.
The OgLuc variant of generation is named and/or about in variant about the amino acid replacement identified in variant Contained escherichia coli cloning is named, and such as, Fig. 6 A shows, in addition to other gained, escherichia coli cloning 16C5 has and puts Change Q20R.
Screening details
The library of gained at expression in escherichia coli and is utilized the system Preliminary screening of automatization and corresponding initiateing OgLuc variant compares and has OgLuc variant (that is, the luminescence increased, the brightness increased or the light increased of the light output increased Launch) or the change of relative specificity.The automatization carried out initially screens as follows: use the single of the library from generation Clone inoculates the minimal medium in the flat board of 96-hole and grows 17 to 20 hours (" M1 cultivation ") at 37 DEG C.Utilize Fresh minimal medium 1: 20 dilution M1 cultivates and cultivates 17-20 hour (" M2 cultivation ") at 37 DEG C.M2 is cultivated 1 : 20 be diluted in inducing culture and grow at 25 DEG C under induction automatically i.e. induction of leaving 17-20 hour (Schagat etc., " KRX Autoinduction Protocol:A Convenient Method for ProteinExpression. " Promega Notes, 98:16-18 (2008)).As novel coelenterazine PBI-3841, PBI-3842, PBI-3857, PBI- 3880, PBI-3881, PBI-3886, PBI-3887, PBI-3897, PBI-3896 or PBI-3894 are used as the end in initial screening During thing, inducing culture contains rhamnose and glucose.When natural coelenterazine, known coelenterazine-h or novel coelenterazine PBI- 3840, when PBI-3889, PBI-3899 or PBI-3900 are used as substrate in initial screening, inducing culture without rhamnose or Glucose.Determine according to the luminescence generated between C1+A4E and novel coelenterazine and use different inducing cultures, i.e. containing Fructus rhamni (Rhamnus davurica Pall.) Sugar and the inducing culture of glucose with and other novel coelenterazine compared with C1+A4E relatively with C1+A4E generation less luminescence Novel coelenterazine be used together.
The cell of the induction of 10 μ L is utilized the HEPES pH8.0 Han 300mM, and 300mM thiourea, 0.3X passively cracks buffering Liquid (" PLB "；Promega Corp. catalog number (Cat.No.) E194A), 60 μ L of 0.3mg/mL lysozyme and 0.003U/ μ L RQ1DNA enzyme split Solve buffer crack, and with containing 150mM KCl, 1mM CDTA, 10mM DTT, 0.5%NP-9(v/v) 50 μ L measure buffer and measure luminescence, and utilize natural, the known or novel coelenterazine of 20 μMs as substrate.Adding Carry out every kind of variant after reagent adding 3 minutes luminous measuring and by corresponding initial about each flat board of relative light units value The meansigma methods of 8 control wells of OgLuc variant is standardized.?Complete in automated system to measure.
Standard sequence methods as known in the art is utilized to check order to identify each institute to OgLuc variant interested State the amino acid replacement of any interpolation in variant.Utilize non-automated (manually) system that variant clone interested is carried out two Level screening.The artificial screening carried out is as follows: by variant clone with three repeated growth in the flat board of 96-hole, and make it express, As about being measured described by the mensuration of automatization, but measure buffer and utilize Multi-channel liquid transfer device manually to add. For each variant, measure luminescence, be averaged and be standardized about corresponding initial OgLuc variant.UtilizeF500 photometer carries out luminous measurement.
Determine the change of relative specificity
By the luminescence of luciferase in the presence of inspection coelenterazine substrate divided by with reference to fluorescence in the presence of coelenterazine substrate The luminescence of element enzyme determines relative substrate specificities.Such as, by the luciferase by the novel coelenterazine utilizing the present invention Luminous divided by utilizing different coelenterazine (such as, natural or known coelenterazine, or the different novel coelenteron of the present invention Element) the luminescence of luciferase determine relative specificity.Test coelenterazine substrate and the reference coelenterazine substrate quilt compared It is considered the comparison substrate pair for determining relative substrate characteristic.
By comparing substrate to testing the relative substrate specificities of luciferase divided by utilizing identical comparison with utilization The relative substrate specificities of the reference luciferase of substrate pair determines the change of relative substrate specificities.Such as, by with The present invention's that different coelenterazine (such as, natural or known coelenterazine or the different coelenterazine of the present invention) compares The relative substrate specificities of the test luciferase of novel coelenterazine divided by from for testing the identical different of luciferase The relative substrate specificities of the reference luciferase of the identical novel coelenterazine of the present invention that coelenterazine compares is to determine phase To specific change.
To utilize the luminescence of a kind of novel coelenterazine compared with the luminescence utilizing different novel coelenterazine.One will be utilized The luminescence of kind natural or known coelenterazine is compared with the luminescence utilizing another natural or known coelenterazine.To utilize The luminescence of a kind of natural or known coelenterazine is compared with the luminescence utilizing novel coelenterazine.
Luminescence (RLU's) of OgLuc variant increases compared with the corresponding initial OgLuc template for novel coelenterazine With luminous the reducing or the change of unchanged expression relative specificity with reference to coelenterazine.Compared with corresponding initial OgLuc The luminous reduction of the OgLuc variant of both coelenterazine of novel and reference, and utilize sending out of the OgLuc variant of novel coelenterazine Light more reduces, and also illustrates that the change of relative specificity.Initial OgLuc with the corresponding coelenterazine for novel and reference The luminous increase of OgLuc variant of comparing shows the raising of activity/stability/expression.If about the novel and coelenteron of reference The two the luminous raising of OgLuc variant of element, and the luminous raising about novel coelenterazine is higher, shows OgLuc variant Increasing and the improvement of activity/stability/expression of relative specificity.
A.C1+A4E variant
Will be previously described in the U. S. application the 12/773,002nd (U.S. Published Application the 2010/0281552nd) C1+A4E (SEQ ID NO:2 and 3) is used as to generate basic homing sequence (that is, the parental generation sequence of the OgLuc variant of other synthesis Row).C1+A4E relative to SEQ ID NO:1 have following amino acid replacement: A4E, Q11R, A33K, V44I, A54F, P115E, Q124K, Y138I and N166R.Utilize the novel coelenterazine (seeing Fig. 4 about example) described in embodiment 1-14 As substrate, the luminescence of the C1+A4E measured containing bacterial lysate as described earlier and with utilize natural and known chamber Intestinal element compares as the luminescence (Fig. 5 A-G) of substrate.Fig. 5 A shows and utilizes natural coelenterazine (" coelenterazine "), known PBI-3880 and novel coelenterazine PBI-3842, PBI-3857, PBI-3881, PBI-3882, PBI-3886 and PBI-3887 make Luminescence for substrate C1+A4E.The luminous area of light of measuring utilizing known and novel coelenterazine is utilized the C1 of natural coelenterazine The emission standards of+A4E also compares multiple and reduces (Fig. 5 B) with natural coelenterazine.Fig. 5 C-E respectively illustrates and utilizes natural coelenteron Plain and novel coelenterazine PBI-3945, the luminescence of C1+A4E of PBI-3894 and PBI-4002.Fig. 5 F shows and utilizes natural chamber Plain and novel coelenterazine PBI-3840 of intestinal, the luminescence of C1+A4E of PBI-3897, PBI-3889, PBI-3899 and PBI-3900. Fig. 5 G show utilize natural coelenterazine, known coelenterazine PBI-3912 and novel coelenterazine PBI-3913, PBI-3925, The luminescence of the C1+A4E of PBI-3939, PBI-3933, PBI-3932, PBI-3946, PBI-3841 and PBI-3896.Data show C1+A4E variant each of may utilize in novel coelenterazine as substrate.
Except as otherwise noted, the C1+A4E variant of generation has the amino acid replacement at least identified in C1+A4E.Logical Cross the library (library 1) of 4400 variant clone of random mutagenesis as described earlier generation C1+A4E and as previously discussed with respect to relatively Specificity changes and/or the carrying out described by improvement of activity change (such as, brightness) is screened.Utilize natural coelenterazine, known Coelenterazine-h (known PBI-3880) and novel coelenterazine PBI-3840, PBI-3841, PBI-3842, PBI-3857, PBI-3881, PBI-3886, PBI-3887, PBI-3889, PBI-3897 and PBI-3900 are as substrate Preliminary screening variant.This Outward, utilize novel coelenterazine PBI-3896 and PBI-3894 as the variant of substrate screening half.Selection comprises and has from screening The flat board of the variant of the sudden change known interested that previous compounds is identified.Separate and close in primary screening test Show the variant of improvement (relative specificity changes or activity change) in one or more tested novel coelenterazine, carry out Order-checking is also screened in two grades of screenings.
In secondary artificial screening, utilize known coelenterazine PBI-3912, coelenterazine-h, coelenterazine-hh, 2-methyl chamber Intestinal element and coelenterazine v；With novel coelenterazine PBI-3840, PBI-3897, PBI-3889, PBI-3899, PBI-3900, PBI- 3925, variant is tested by PBI-3944, PBI-3932, PBI-3945, PBI-3913 and PBI-3896 as substrate.Figure 6A-D summarize variant about the standardized average luminescence of C1+A4E (" clone ").Fig. 6 A-D summarizes putting in these variants Change (" aminoacid sequence "), its have at least one in the amino acid replacement of following interpolation: A14V, G15R, Q18L, Q20R、L22I、E23K、L27V、L27M、K33N、T39I、E49K、F54S、F54I、D55G、I56V、V58I、V58L、I59T、 S66T、G67S、F68S、L72Q、M75K、I76N、F77T、F77C、K89E、I90V、I90T、L92H、H93R、M106K、Y109F、 P113T, I117F, T126R, V127A, L136M, D139G, P145L, S148T, C164S or A169V.
As shown in figures 6 a-c, the amino acid replacement of the 54th, 92 and 109 is interested, because the putting of these positions Change and provide the output of higher light or the relative specificity of improvement, i.e. specificity and natural coelenterazine gap are remote and close at least A kind of novel coelenterazine (as shown in figures 6 a-c).Amino acid replacement F54I in clone 29H7 utilizes natural coelenterazine and novelty Some in coelenterazine provide the output of higher light.Amino acid replacement Q18L in clone 40H11, the ammonia in clone 04A12 Base acid is replaced the amino acid replacement Y109F in L92H, and clone 43F9 and is provided the relative specificity of improvement.
What table 5 listed generation as described earlier has other amino acid replacement (" ammonia at the 77th, 92 or 109 Base acid change ") C1+A4E variant.The light the increased output analyzing these variants as described earlier, i.e. utilize natural chamber Intestinal element, it is known that coelenterazine-hh and novel coelenterazine PBI-3939, PBI-3894, PBI-3896, PBI-3897, PBI-3932 Or PBI-3925 is as the substrate screening variant than C1+A4E at least 1.3 × bright.Following other displacement produces compares C1+A4E At least 1.3 × bright variant: L92G, L92Q, L92S, L92A, L92M, L92H, L92Y, F77W, F77Y, F77S, F77T, F77V, F77A, F77G, F77C, F77D, F77M and Y109F.As shown in table 5, L92H, F77W and F77A displacement utilizes PBI- 3897, PBI-3896 and PBI-3932 has and the most significantly improves.
The site that table is 5: the 77,92 and 109 is saturated
L92Q 2 1.8 1.6 1.3 1.4 2.8 14 3.4
L92S 2.9 1.5 2.9 2.7 6
L92A 2.5 1.3
L92M 1.3
L92H 2.2 21 9.1 3.4 5.9
L92Y 2.5
F77W 1.4 1.4 1.4 8.3 3.2 1.7 2.3
F77Y 1.6 1.3 4.9 6.5
F77S 2.6
F77T 2.3
F77V 2.3
F77A 7.9 2.5
F77G 3.1
F77C 2.3
F77D 1.5
F77M 1.5 1.6
Y109F 1.34 14
Other C1+A4E variant (A group) is generated with at following amino acid position as described earlier by direct mutagenesis At least one in relative to SEQ ID NO:1 there is other displacement: 18,20,54,59,72,77,89,92,109,113, 127,136 or 164.These amino acid positions are selected to be because, primary screener based on library 1 and two grades of screenings, these positions The displacement at place utilizes at least one in below to have the overall light increased as substrate with C1+A4E compared with and exports: novel cavity Intestinal element PBI-3841, PBI-3896, PBI-3897, PBI-3894, PBI-3925 or PBI-3932 or known coelenterazine 2-first Base coelenterazine or PBI-3912.Fig. 7 lists variant (" clone ") contained in each variant and other amino acid replacement.As Previously described as about described by two grades of artificial screenings with three replication variant clone and about C1+A4E standard Change.Fig. 8 A-B and 9 shows that variant listed in Fig. 7 utilizes different coelenterazine as the standardized average luminescence of substrate. Fig. 8 A-B and 9 shows the luminous bigger increase of new compound relatively listed compared with C1+A4E or compared with C1+A4E The luminous nothing of more known coelenterazine changes or reduces.Clone QC27, its have other amino acid replacement Q18L, F54I, L92H and Y109F, utilizing the luminescence of PBI-3896 to have 561.32 multiples compared with C1+A4E increases, and utilizes PBI-3894 to have There are 392.98 multiples to increase, and utilize PBI-3896 to have 283.85 multiples increases.The data show Q18L, L92H and Y109F can be combined with each other and have other displacement to produce the variant of the relative specificity with improvement.
By other Permutations combination interested identified from library 1 to generate other variant (B group) (Figure 10).Following At least one in amino acid position carries out other amino acid replacement relative to SEQ ID NO:1: 18,20,54,71,77, 90,92,109 or 127.These displacements show utilize at least one in following novel coelenterazine as the improvement of substrate: PBI-3841, PBI-3896, PBI-3897, PBI-3894, PBI-3925 or PBI-3932.Utilize natural coelenterazine, known Coelenterazine-hh and novel coelenterazine PBI-3939, PBI-3945, PBI-3840, PBI-3932, PBI-3925, PBI-3894 and PBI-3896 is as about being measured these variants described by A group variant.As described earlier as artificial about two grades Described by screening with three replication variant clone and about C1+A4E standardization.Figure 11 shows and utilizes different coelenterons Element is as the standardized average luminescence of the variant listed by Figure 10 of substrate.Figure 11 shows compared with C1+A4E relatively listed new The luminous substantial increase of type coelenterazine or compared with C1+A4E the luminescence of more natural and known coelenterazine unchanged or Reduce.
Other amino acid replacement I90V and/or Y109F is utilized to generate other variant (C group) and give birth to from A group or B group The variant become compares (seeing Figure 12).Utilize natural coelenterazine, known coelenterazine-hh and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, PBI-3840, PBI-3925, PBI-3932, PBI-3894, PBI-3896 and PBI-3897 conduct Substrate utilization has I90V displacement (" I90V "), Y109F displacement as comprised about the mensuration described by A group recombinant The clone of the variant of (" Y109F ") or two kinds of displacements (" LE2 ") is compared with clone QC#27, QC#2E7, QC#2F4 and QC#1A11 Relatively (Figure 12).As described earlier as about described by two grades of artificial screenings with three replication variant clone and about C1+A4E standardization (Figure 12).Figure 12 show compared with C1+A4E relatively listed novel coelenterazine luminous substantial increase and More natural or known coelenterazine luminous unchanged or reduce compared with C1+A4E.Figure 12 shows that I90V is natural chamber Some in intestinal element and novel substrate provide higher light output.
B.QC27 variant
As described earlier by the change with other amino acid replacement Q18L, F54I, L92H and Y109F from A Body QC27 (SEQ ID NO:4 and 5) is cloned in the carrier that pF4A modifies and melts with structure C-end HT7 (Promega Corp.) Hop protein (" QC27-HT7 ") (SEQ ID NO:44 and 45).As described earlier by random mutagenesis generation QC27-HT7's 4400 variants (library 2), and utilize natural coelenterazine and novel coelenterazine PBI-3896 and PBI-3897 as substrate as first Changing the relative specificity increased described by before carries out primary screener.Utilize natural coelenterazine, known coelenterazine-hh and Novel coelenterazine PBI-3897, PBI-3896 and PBI-3894 select variant clone as substrate in two grades of artificial screenings, enter Row order-checking and mensuration.
Figure 13 lists the other amino acid replacement (" sequence ") identified in these variants (" sample "), and at two grades Screening utilizes natural coelenterazine, it is known that coelenterazine-hh and novel coelenterazine PBI-3897, PBI-3896 and PBI-3894 make The standardized luminescence of initial QC27-HT7 about correspondence for substrate variant.Variant in Figure 14, has following other At least one in amino acid replacement: F1I, R11Q, L18I, L18Q, V21L, V21M, L22F, F31I, Q32H, V45E, L46Q, S47P、G48R、E49D、G51E、D55E、G67S、F68Y、F68L、Q69H、L72Q、E74K、E74I、M75K、I76F、I76V、 H86R、I90T、H92Q、H92R、T96A、V98F、I99V、I99T、V102M、M106I、F109Y、L142V、V158I、T159S、 L168F or G170R (G170R joint area between HT7 and OgLuc variant).
M75K in amino acid replacement F68Y in variant 24B12, the L72Q in variant 29C4 and variant 3H11 is respectively sky Some in right coelenterazine and novel substrate provide higher light output.Amino acid replacement V21L in variant 25A11 and change H92R in body 1B6 provides the relative specificity increased.These two kinds of displacements are to utilize novel coelenterazine to believe as substrate luminescence Number reduce, but utilize natural and known coelenterazine to reduce more situation as substrate.
Direct mutagenesis is utilized to generate other QC27-HT7 variant to have specific aminoacid as described earlier Displacement (Figure 14).At least one in the following amino acid position relative to SEQ ID NO:1 generates other displacement: 21,68,72,75,76,90,92 and 158, the increasing that relative specificity as shown in Figure 14 changes due to these position display High.Figure 15 shows and utilizes natural coelenterazine, known coelenterazine-hh and novel coelenterazine PBI-3897, PBI-3841, PBI- 3896 and PBI-3894 as substrate QC27-HT7 variant about the corresponding standardized luminescence of initial QC27-HT7.Such as Figure 15 Seen in, by combined with V158I to three kinds of amino acid replacements F68Y, L72Q and M75K, as shown in variant QC27#1, right The output of higher light is provided in every kind of tested coelenterazine.
C.QC27-9a variant
Use from the variant QC27-9a (SEQ ID NO:6 and 7) of B, i.e. have other amino acid replacement V21L, The QC27-HT7 fusion protein of H29R, F68Y, L72Q, M75K and V158I, generates library as homing sequence.Such as previous institute Describe generates 4400 variants (library 3) of QC27-9a by random mutagenesis and utilizes natural coelenterazine and novel coelenterazine The relative specificity that PBI-3841 and PBI-3897 screening is increased changes.Utilize natural coelenterazine, it is known that coelenterazine-hh, Coelenterazine-the h known and novel coelenterazine PBI-3841 and PBI-3897 as substrate as described earlier at two grades of artificial sieves Choose selection variant clone, check order and measure.Figure 16 lists the other displacement identified in variant (" sample ") (" amino acid change "), and two grades screening in utilize natural coelenterazine, known coelenterazine-hh, it is known that coelenterazine-h and Novel coelenterazine PBI-3841 and PBI-3897 are as the average luminescence about corresponding initial QC27-9a of substrate variant.Phase To specific increase to represent utilize the luminous fall of novel, natural and known coelenterazine variant compared with starting template Low, but utilize natural reducing with known coelenterazine luminescence to obtain more situation.Such as, there is the change of amino acid replacement L22F Body 30D12 utilizes the loss of activity that novel coelenterazine PBI-3841 and PBI-3897 have about three times.But, utilize natural coelenteron Element, it is known that coelenterazine-h and known coelenterazine-hh, the luminescence of variant 30D12 reduces 10 times or more.
Figure 17 shows and utilizes natural coelenterazine, it is known that coelenterazine-hh and novel coelenterazine PBI-3841 and PBI- 3897 as substrate belong to luciferase (referred to herein as " hRL ") (SEQ ID NO:30 and 31) with humanized sea pansy compared with C1 + A4E, the luminescence of QC27-HT7 and QC27-9a.Although utilizing the reaction Billy PBI-3841 of the QC27-9a of PBI-3897 QC27-9a brighter (seeing Figure 17), evolving trend, i.e. the intensity of luminous increase, is the highest (table 6) about PBI-3841. By combined to luminous increase (440 times) and the luminous reduction (800 times) of natural coelenterazine, indicate and natural coelenterazine phase It is compared with the change (350,000 times) of the relative specificity of PBI-3841QC27-9a.
Table 6: about the OgLuc variant of PBI-3897 and PBI-3841 compared with the natural coelenterazine and coelenterazine-hh The change of relative specificity
D.IVY variant
As described earlier by IVY (SEQ ID NO:8 and 9), have other amino acid replacement F54I, I90V and To build C-end HT7 fusion protein (" IVY-HT7 ") in the carrier that the C1+A4E variant clone of F77Y is modified to pF4A.Pass through Random mutagenesis generates 4400 variants (library 4) of IVY-HT7 and utilizes natural coelenterazine, it is known that coelenterazine-hh and novel The light output that coelenterazine PBI-3840, PBI-3889, PBI-3925, PBI-3932 and PBI-3945 increase as substrate screening (that is, the brightness increased) and the relative specificity increased.Utilize natural coelenterazine, it is known that coelenterazine-hh and novel coelenterazine PBI-3889, PBI-3939, PBI-3945 and PBI-4002 as substrate as described earlier two grades screening in three Repeat to select variant clone, check order and measure.Figure 18 and 19 lists other the putting identified in variant (" sample ") Change (" amino acid change ") and have selected natural coelenterazine at two grades of sieves, it is known that coelenterazine-hh and novel coelenterazine PBI- 3889, PBI-3939, PBI-3945 and PBI-4002 are as the average luminescence about IVY-HT7 of substrate variant.Figure 18 arranges Having gone out according to those variants (A group) utilizing the performance of PBI-3945 to select, it has at least in following amino acid replacement Kind: Q18H, D19N, Q20P, Q32P, K33N, V38I, V38F, K43N, I44F, E49G, I60V, Q69H, I76N, Y77N, Y94F, G95S, G95D, F110I, V119M, K124M, L149I or R152S.Figure 19 lists according to the table utilizing PBI-3889 Those variants (B group) now selected, its have at least one in following amino acid replacement: F6Y, Q18L, L27V, S28Y, Q32L, K33N, V36E, P40T, Q42H, N50K, G51R, H86L, N135D or I155T.
Direct mutagenesis is utilized to generate the other IVY-HT7 with other amino acid replacement as described earlier. 19,20 Figure 20 lists the variant of at least one having in amino acid position additionally below relative to SEQ ID NO:1:, 27,32,38,43,49,58,77,95,110 and 149, because these displacements are identified in the variant of Figure 18, which show and connect The specificity of nearly PBI-3945 and PBI-4002.Figure 21 shows that variant listed in Figure 20 utilizes natural coelenterazine, it is known that Coelenterazine-h, it is known that coelenterazine-hh and novel coelenterazine PBI-3939, PBI-3945, PBI-4002, PBI-3932 and PBI- 3840 as substrate about the standardized luminescence of IVY-HT7.Show without variant and surmount increasing of IVY-HT7, but there is example Son such as variant C5.19 (SEQ ID NO:12 and 13) its utilize the luminescence of natural or known coelenterazine to reduce about 3-4log, but utilize the luminescence of PBI-3945 and PBI-4002 to reduced by only twice.Variant C5.19 has other aminoacid and puts Change L27V, V38I and L149I.
6,18 Figure 22 lists at least one of amino acid position relative to SEQ ID NO:1 with following increase:, 27,28,33,34,36,40,50,51,135 and 155, because these displacements are identified in the variant of Figure 19, which show and connect The specificity of nearly PBI-3889 and PBI-3939.Figure 23 shows that variant listed in Figure 21 utilizes natural coelenterazine, it is known that Coelenterazine-hh and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, PBI-4002, PBI-3932 and PBI-3840 make For substrate and about the standardized luminescence of IVY-HT7.The luminous reduction of more every kind of variant compared with IVY-HT7.Utilize PBI- Variant C1.3 (SEQ ID NO:10 and the 11) Billy of 3939 with natural or known coelenterazine have about 2000 times higher Luminous.Variant C1.3 has other amino acid replacement F6Y, K33N, N135D and I155T.
The optimal IVY-HT7 variant that relative specificity changes compared with hRL and IVY-HT7 is C5.19, and it utilizes PBI- 3945 have optimal luminescence, and C1.3, and it utilizes PBI-3889 to have optimal luminescence.Figure 24 shows and utilizes natural chamber Intestinal element, it is known that coelenterazine-h, it is known that coelenterazine-hh and novel coelenterazine PBI-3939 and PBI-3945hRL, IVY-HT7, C5.19 (C-end HT7 fusant) and the luminescence of C1.3 (C-end HT7 fusant).
E.IV variant
Generate IV (SEQ ID NO:14 and 15) as described earlier, i.e. have other amino acid replacement F54I and The C1+A4E variant of I90V.For determine as transcribed reporter the brightest variant, utilize natural coelenterazine, it is known that coelenterazine C1+ is measured as described earlier as substrate with novel coelenterazine PBI-3939, PBI-3945, PBI-3889 and PBI-4002 The luminescence that A4E (SEQ ID NO:2 and 3), IVY (SEQ ID NO:8 and 9) and IV (SEQ ID NO:14 and 15) provide.HRL uses Compare.As seen in Figure 25, IV is brighter than C1+A4E and IVY.Amino acid replacement F54I in IV is for natural coelenteron Some in element and novel substrate provide the output of higher light.The coelenterazine all three variant utilizing test is brighter than hRL.
Comprehensively from A, B and D (that is, the screening in the library generated from C1+A4E, IVY and the QC27 as homing sequence) The light that data increase to determine with multiple coelenterazine to have exports those other aminoacid of (that is, the brightness increased) and puts Change.Generation as described earlier has specific other the putting of the reduction natural coelenterazine to twice to ten times The IV variant changed.As listed by Figure 26, IV variant (" clone ") has the another of at least one in following amino acid replacement Outer amino acid replacement (" sequence "): F1I, E4K, Q18L, L27V, K33N, V38I, F68Y, M70V, L72Q, M75K or V102E。
The variant clone of 16 flat boards of whole combinations of amino acid replacement is carried out primary screener and utilizes natural coelenteron Element, it is known that coelenterazine-h, it is known that coelenterazine-hh and novel coelenterazine PBI-3889 and PBI-3945 as substrate as previously The described robotic method utilizing automatization is measured.Artificial screening is utilized to repeat choosing with three as described earlier Select the luminous variant with improvement, check order and measure.Utilize natural coelenterazine, it is known that coelenterazine-h, it is known that chamber Intestinal element-hh and novel coelenterazine PBI-3889, PBI-3939, PBI-3945 and PBI-4002 measure luminescence as substrate.Use Corresponding homing sequence IV and hRL is as comparison.
Figure 26 lists variant, and the other amino acid replacement identified in variant.Figure 27 shows in two grades of screenings Variant about the standardized average luminescence of IV.Variant 8A3 (SEQ ID NO:26 and 27), it has other amino acid replacement F1I, L27V and V38I, utilize novel coelenterazine to have the relative specificity increased, but be not as brighter as IV.Variant 8F2 (SEQ ID NO:46 and 47), it has other amino acid replacement L27V, and 3 in 4 novel coelenterazine used by utilization provide increasing High relative specificity and brightness.Variant 9B8 (SEQ ID NO:18 and 19), its have other amino acid replacement Q18L, F68Y, L72Q and M75K, brighter than all substrates and additionally provide some the relative specificity advantages surmounting natural coelenterazine.Become Body 9F6 (SEQ ID NO:20 and 21), it has other amino acid replacement Q18L, L27V, V38I, F68Y, L72Q and M75K, Show and utilize similar increasing seen by 8F2.Variant 15C1 (SEQ ID NO:16 and 17), it has other aminoacid and puts Changing E4K, K33N, F68Y, L72Q and M75K, it is brighter for whole novel coelenterazine, but does not have the relatively special of any raising Opposite sex benefit.In the case of IV, the amino acid replacement Q18L in variant 1D6 provides the relative specificity increased, i.e. with sky So coelenterazine gap is remote and close to novel substrate.In a word, amino acid replacement L27V provides relative specificity in the case of IV.
Figure 28 shows compared with IV and hRL, utilizes natural coelenterazine, it is known that coelenterazine-hh, it is known that coelenteron Element-h and novel coelenterazine PBI-3939, PBI-3945, PBI-3889 and PBI-4002 as 8A3 in substrate postsearch screening, The luminescence of 9B8,9F6 and 15C1.Compared with utilizing natural coelenterazine variant 8A3 with IV, brightness has 2log reduction.Utilize natural Compared with coelenterazine variant 9F6 with IV, brightness has 1log reduction.The variant 15C1 utilizing PBI-3945 is the brightest, but signal Half-life short (seeing embodiment 27).
F.9B8 variant
9B8 variant from E is modified further and there is the light emission increased to generate and/or PBI-3939 is increased The other variant of relative specificity.Amino acid replacement L72Q shows as the favourable of the light emission (that is, brightness) for increasing Amino acid replacement because identifying this displacement in variant 9B8,9F6 and 15C1, it all shows the light emission increased.For Determine whether other amino acid replacements of the 72nd will provide similar brightness to increase, by making the 72nd with interchangeable residue The other variant of saturated generation 9B8 as described earlier.Prepare four of E. coli lysate repeat and utilize PBI- 3939 are analyzed about brightness as described earlier as substrate, but except for the difference that measure buffer containing 10mM CDTA, 150mM KCl, 10mM DTT, 100mM HEPES, pH7.0,35mM thiourea and 0.5%NP-9(v/v).Table 7 list by about as indicated in the standardized luminescence of 9B8 (" RLU (about 9B8 standardization) "), having compared with 9B8 There is luminous 9B8 variant (" variant ") that is similar or that increase, i.e. multiple increases.Amino acid replacement A, G, N, R of 72nd and M provides the brightness benefit the most identical with aminoacid Q, i.e. 1 times.
Table 7: there is compared with variant 9B8 similar luminous variant.
Variant RLU (about 9B8 standardization)
9B8+Q72A 1.1
9B8+Q72G 1
9B8+Q72N 1
9B8+Q72R 1
9B8+Q72M 1
By making the 18th, 68,72,75 and 90 amino acids in variant 9B8 saturated, generate newly as described earlier Type PBI-3939 has the other variant of the relative specificity increased.Prepare E. coli lysate and utilize natural coelenterazine It is analyzed about brightness as described earlier as substrate with novel PBI-3939.Emission standards about corresponding 9B8 Change, determine relative special from the luminescence of variant utilizing PBI-3939 with the luminous ratio of the variant utilizing novel coelenterazine Property.Table 8 lists the 9B8 variant (" variant ") having at least 1.1X times relative specificity increased of PBI-3939.Result Show that at least one the other change in each site is compared natural coelenterazine and provided relative specificity for PBI-3939. The highest multiple at the 18th the 9B8 variant with amino acid replacement K, D, F, G, Y, W and H with relative specificity increases.
Table 8: there is the variant of the relative specificity increased for PBI-3939
G.9B8+K33N variant
Generating other variant, 9B8opt+K33N (SEQ ID NO:42 and 43) is to study amino acid replacement K33N pair Benefit in brightness, relative specificity and heat stability.Check 9B8opt+K33N in different applications and compared with 9B8opt Relatively (described in embodiment 25A).
Preparation is containing the E. coli lysate of variant 9B8opt or 9B8opt+K33N and carries out as described earlier point Analysis, except for the difference that measures buffer containing 0.1%NP-9(v/v).Utilize novel PBI-3939 and natural coelenteron Element measures the luminescence generated from lysate as substrate.The variant that calculates as described earlier is for PBI-3939 and natural coelenteron The relative specificity of element.9B8opt+K33N (" K33N ") has light more higher than natural coelenterazine output compared with 9B8opt (RLU) and relative specificity higher for PBI-3939 (Figure 29), show that K33N displacement provides the output of higher light and increases High relative specificity.
9B8opt+K33N is utilized to construct novel OgLuc library as starting template.Utilize PCR Random Mutagenesis Kit(ClonTech；Catalog number (Cat.No.) #630703) construct random library.Employ condition 5 (as listed by user's manuals) generate other variant, by by the sequence data from 83 clones randomly choosed Calculate and averagely sport the 2.6 every genes of sudden change.As described earlier by this PCR library clone to non-fused based on pF4Ag Vector background and sandwich background in, i.e. Id-OgLuc-HT7 (described in embodiment 45).Based on pF4Ag non-melt Close vector background named (NF).Variant in sandwich vector background is named (F).For PCR primer being cloned into two kinds of loads In body, by aminoacid, i.e. glycine, it is attached in the variant sequence thereof in pF4Ag, OgLuc variant generates new 170 (" 170G ").This 170G is present in sandwich construct, but is considered as between OgLuc and HT7 in this case The part of joint.For each library, measure 4,400 escherichia coli clonings as described earlier divided by lower difference.Cracking Buffer contains 300mM MES pH6.0 rather than HEPES, and 0.5%NP-9 (v/v), but without thiourea. Measure buffer and contain 100mM MES pH6.0 rather than HEPES, and 35mM thiourea.This mensuration volume is as follows: 10 μ L cells, 40 μ L Lysis buffer and 50 μ L measure buffer.
About having increase luminous other compared with 9B8opt+K33N+170G (SEQ ID NO:68 and 69) Variant screens the PCR library in nonfused background based on pF4Ag.Then measure in HEK293 cell and NIH3T3 cell Selected variant.For every kind of cell type, inoculate 15,000 cell and at 37 DEG C overnight growth.Second day, utilize The 10ng pGL4.13 (Promega Corp.) transfectional cell as described in embodiment 25 is as transfection control, and uses 100ng OgLuc test DNA transfectional cell.Remove culture medium, and the lysis buffer with 100 μ L as described in embodiment 25 splits Solve cell, but except for the difference that lysis buffer contains 100mM MES pH6.0 rather than HEPES, and utilize Luminometer measures luminescence.For each sample, the 50 μ L lysis buffers containing 20 μMs of PBI-3939 are utilized to measure 10 μ L Lysate.For transfection control, with the BRIGHT-GLO of 50 μ LTMMeasure reagent and measure the lysate of 10 μ L.
Table 9 shows that the luminous multiple of the variant in escherichia coli, HEK293 and NIH3T3 cell increases and in variant The amino acid replacement found.Variant 27A5 (NF) (SEQ ID NO:70 and 71), 23D4 (NF) (SEQ ID NO:72 and 73) and 24C2 (NF) (SEQ ID NO:74 and 75) has at least 1.3 multiplications of luminescence in Bacillus coli cells and HEK293 cell Add.
Table 9: in Bacillus coli cells, HEK293 cell and NIH3T3 cell compared with 9B8opt+K33N+170G by The luminous increase that OgLuc variant is generated
Based on data above, design also generates in the environment without the non-fused vector background based on pF4Ag of 170G Other combinatory variants.Variant is analyzed as described above in Bacillus coli cells, HEK293 cell and NIH3T3 cell And compared with 9B8opt+K33N.Checked the luminescence of the variant utilizing natural coelenterazine.Table 10 shows that escherichia coli are thin The luminescence of variant in born of the same parents, HEK293 cell and NIH3T3 cell, and the amino acid replacement that in variant, (" sample ") finds.By inciting somebody to action Other amino acid replacement in variant adds prefix " 9B8opt+K33N. " to and names variant.Table 11 shows at large intestine bar Relative for PBI-3939 of bacterium cell, NIH3T3 cell and variants more different compared with natural coelenterazine in HEK293 cell Specificity.As shown in Figure 10, variant 9B8opt+K33N+T39T+K43R+Y68D (" V2 "；SEQ ID NO:92 and 93) greatly Enterobacteria has the luminescence increased and there is in NIH3T3 cell slightly increasing of luminescence.In three the cell classes checked In type, variant 9B8opt+K33N+L27V+K43R+Y68D (" L27V, K43R, Y68D ") has the neutral of luminescence and increases (table 10) and surmount 9B8opt+K33N 5X multiple increase relative specificity (table 11).
Table 10: in Bacillus coli cells, NIH3T3 cell and HEK293 cell compared with 9B8opt+K33N by The luminous increase that OgLuc combinatory variants generates
Table 11: Bacillus coli cells, NIH3T3 cell with in HEK293 cell compared with natural coelenterazine OgLuc group Close the variant change for the relative specificity of PBI-3939
Generate containing at least the amino acid replacement additionally below relative to SEQ ID NO:1 from 9B8opt+K33N The other OgLuc variant planted: L27V, T39T, K43R, Y68D or S66N (see table 12 about the amino acid replacement in variant In " sample ").By adding the other amino acid replacement in variant to prefix " 9B8opt+K33N. " named later Variant.Check from the other variant of the above and variant 9B8opt+K33N+L27V+Y68D (" L27V, Y68D "), 9B8opt+K33N+L27V+K43R+Y68D (" L27V, K43R, Y68D "), 9B8opt+K33N+L27V+K43R+S66N (" L27V, K43R, S66N ") and 9B8opt+K33N+T39T+K43R+Y68D (" T39T, K43R, Y68D "；Also referred to as " V2 ") Brightness, relative specificity, signal stabilization and heat stability.By variant and variant 9B8opt (" 9B8 ") and 9B8opt+K33N (" K33N ") compares.
Preparation contains the E. coli lysate of variant and is analyzed as described earlier.Utilize novel PBI-3939 and Natural coelenterazine measures the luminescence generated from lysate as substrate.The emission standards that the luminescence of variant is generated about 9B8opt Change (table 12).By PBI-3939 being utilized divided by utilizing the change as substrate of the natural coelenterazine as the luminescence of the variant of substrate The luminous of body calculates the variant relative specificity (table 12) for PBI-3939 and natural coelenterazine.These data show that aminoacid is put Change L27V and reduce the specificity for natural coelenterazine.
Table 12: the luminous increase that generated by OgLuc variant compared with 9B8 in bacterial lysate and with natural coelenteron Element compares the specific change for PBI-3939 of the OgLuc variant
Sample Multiple more than 9B8 Exceed the multiple of coelenterazine
9B8 1.0 7
K33N 1.1 21
T39T, Y68D 0.9 12
T39T, L27V, K43R 1.2 149
L27V, T39T, K43R, Y68D 1.8 110
T39T, K43R, Y68D 1.6 21
L27V, T39T, K43R, S66N 1.3 114
L27V, K43R, Y68D 1.3 110
L27V, Y68D 1.0 63
L27V, K43R, S66N 1.1 114
H.V2 variant
V2 is utilized to devise as template (having the 9B8opt of other amino acid replacement K33N+T39T+K43R+Y68D) A set of other variant.Based on 1) according to multiformity known to the comparison based on structure of 28 kinds of fatty acid binding proteins (1VYF, 1FDQ、2A0A、1O8V、1BWY、2ANS、1VIV、1PMP、1FTP、2HNX、1JJJ、1CBR、2CBS、1LPJ、1KQW、2RCQ、 1EII、1CRB、1IFC、2PYI、2JU3、1MVG、2QO4、1P6P、2FT9、1MDC、1O1U、1EIO；See U.S. Published Application No. 2010/0281552), or 2) the optional residue of the position previously identified is worked in substrate specificity Detect and devise the displacement shown in table 13.Change listed under " consensus sequence " in table 13 relates to through comparison, above-mentioned Fatty acid binding protein at least 50% in the residue identified.Change listed under " main minority " relates to above-mentioned The majority of fatty acid binding protein is identified but is found in the residue of less than the 50% of aligned sequence.Listed changing under " other " Become and relate to the residue lower than the main minority residue frequencies of position given in the sequence of alignment.Finally, " special Property " under listed change relate to speculating and participate in determining that variant has for the specific position of coelenterazine or coelenterazine analog Close.Such as, specificity designed by (leucine residue in parental sequences, i.e. V2) is changed into and is become other and dredge at the 27th The aminoacid of the chemistry that aqueous residue or representative substitute (such as, comprises the hydrophobic residue of ring, comprises without charge polarity side chain Residue, or comprise the residue of charged side chain)；With the 40th at specificity designed by (proline in parental sequences) change (chemistry (such as, comprises other hydrophobic residue of ring, comprises without charge polarity side chain to become the selection to different chemistry Residue or comprise the residue of charged side chain)；Notice that glycine, glutamine, isoleucine and leucine are in this position It is accredited as the fatty acid binding protein of alignment).
Table 13
The site directed mutagenesis protocol (seeing previous example) utilizing standard builds variant, and by the Plastid transformation of gained to big For analysis in enterobacteria.Make cultivation raw according to the induction of leaving of standard in minimal medium as described earlier Long.Successively add in the Bacillus coli cells of the conversion of the cultivation of 10 μ L 40 μ L lysis buffers (100mM MES pH6.0, 0.3X PLB, 0.3mg/mL lysozyme, 0.003U/ μ L RQ1DNA enzyme I and 0.25%NP-9 (v/v)) and Mensuration buffer (1mM CDTA, 150mM KCl, 2mM DTT, 20 μMs of PBI-3939 or natural coelenterons of equal-volume (50 μ L) Element, 100mM MES pH6.0,35mM thiourea and 0.5%NP-9(v/v)).? Luminescence is measured on 96Microplate Luminometer (Promega Corp.).
Table 14 summarizes the different amino acid replacement identified in analysis.Data are rendered as closing about Parental clones (V2) In for the emission standards measured by PBI-3939 and natural coelenterazine.Also show the specificity to PBI-3939 about sky So relative changes of coelenterazine.
Table 14
I.L27V variant
Utilize OgLuc variant L27V as starting template, i.e. homing sequence or parental sequences, generate other variant, Wherein more amino acid whose (table 15) in L27V variant is replied as the natural OgLuc luciferase at SEQ ID NO:1 The aminoacid of middle discovery.Variant is built as described earlier by direct mutagenesis.Utilize natural the most as described earlier Coelenterazine or the relative activity of PBI-3939 screening variant.After adding substrate/mensuration reagent (as described in H) 5 MinuteF500 is upper to be measured luminous and is standardized about L27V variant starting template.Split The SDS-PAGE solving thing analysis shows similar expression (data are not shown).
Table 15 shows and utilizes natural coelenterazine or the relative activity of PBI-3939L27V variant.Relative activity ＜ 1 shows It is harmful for replying compared with the residue of this site in L27V variant.Relative activity ＞ 1 show with in L27V variant The residue in this site compares and replys for activity is favourable.Below the other tables of data of some of these mutants understands: Examine the heat stability of 166K, 54F, 54A and L27V.The T of 166K, 54F and 54A1/260 DEG C are respectively 87,74 and 33%, table These displacements bright result in the reduction of heat stability.The Km value of these 4 identical variants is following: for natural coelenterazine, L27V is 16 μMs, and 54A is 23 μMs, to be 40 μMs and 166K be 21 μMs to 54F；For PBI-3939, L27V be 18 μMs, 54A be 62 μMs, It is 23 μMs that 54F is 163 μMs and 166K.This demonstrate the higher substrate affinity of L27V, especially with respect to the displacement of the 54th.
Table 15
The mutation analysis that embodiment 23 is the 166th
A. it is the different aminoacid evaluating the 166th effect to uciferase activity, exists as described earlier By the arginine of the 166th (that is, in the environment of wild type OgLuc sequence SEQ ID NO:1) in the environment of pF4Ag carrier (R) each during residue substitutions is other 19 aminoacid.Then as described earlier expression in escherichia coli these 166th variant.
For building lysate, by 50 μ L0.5X FASTBREAKTMCell lytic reagent (Promega Corp.) adds to In the cultivation of the induction of 950 μ l, and mixture is hatched 30 minutes at 22 DEG C.In order to analyze, utilize 100 μMs of PBI-3939, 30 μMs of natural coelenterazine or 22 μMs of coelenterazine-h survey in the mensuration reagent (as previously described in embodiment 22H) of 50 μ L The lysate of fixed 50 μ L.Measure luminescence (Figure 30 A-C) as described earlier.Figure 30 A-C shows the relative of N166 mutant Specificity.Western blot analysis determines the similar expression (data are not shown) of whole variant.
B. have evaluated in wild type OgLuc or N166R background specific individually amino acid replacement L27V, A33N, K43R, M75K, T39T, L72Q and F68D.Single amino acid replacement is generated as described earlier by direct mutagenesis, as Previously described at expression in escherichia coli and utilize by 22 μMs of natural coelenterazine that to measure reagent measurings luminous (as previously in reality Execute described in example 22H) (Figure 30 D).Western blot analysis determines the similar expression of all variants, and (data are not shown Go out).
Embodiment 24 deletion mutants
According to following to L27V variant generation disappearance:
Table 16
Disappearance # The disappearance generated
27 Residue 1-27 and Val-1
52 Residue 1-52 and Val-1
64 Residue 1-64 and Val-1
84 Residue 1-84 and Val-1
19 Residue 65-83
23 Residue 65-87
23A1 Residue 65-87+G64D
The N-end of OgLuc variant L27V is methionine, valine and phenylalanine, i.e. MVF.For the purpose of numbering, recognize Being the first aminoacid for phenylalanine, the valine in " Val-1 " expression " MVF " is lacked.The methionine of " MVF " is contained in this In a little disappearances.L27 deletion mutants is cloned in pF4Ag carrier expression in escherichia coli KRX cell as described earlier. Carry out induction as described and be prepared by lysate, utilize mensuration reagent (previously described；100 μMs of PBI-3939) analyze Lysate, and side as described earlier amount is luminous (Figure 31).Data prove that the less fragment of OgLuc variant also can produce and send out Light.
Embodiment 25 OgLuc variant codon optimized
A.IV and 9B8
IV and 9B8OgLuc variant is used as codon optimized template.As those skilled in the art understands , it is therefore an objective to two-sided: 1) remove the regulation and control that may can interfere with OgLuc variant or the known transcription factor binding site expressed Point or other regulating and controlling sequences, such as, promoter component, donor splicing site/acceptor site, montage silencer, Kozak sequence and poly-gland Nucleotide signal, and 2) change DNA sequence (silent mutation via not changing protein sequence) to eliminate seldom used codon, and partially The most frequently used codon in the cell of good escherichia coli, the mankind, other mammals or other most eukaryotes (Wada etc., Nucleic Acids Res., 18:2367 (1990)).
Each its variant designs is referred to as to the optimization of opt (also known as optA) and the two of optB different IV and 9B8 Sequence.By each site is identified two kinds optimal, the most modal human codon (seeing table 17) and the most randomly Select alternative one to have mixed to each its variant designs the sequence optimized, i.e. opt/optA in each site.For optB Form, previous, the form of the optimization that codon uses, i.e. opt/optA, as starting template, and each codon is closed Another in two optimal human codon that this codon optimizing strategy is identified is replaced.As an example, IV sequence Or the leucine of the 3rd in 9B8 sequence is by coded by codon TTG.TTG be not in human cell leucic two kinds One of common codon, and therefore this codon is become replacement, leucine codon more often, CTC (opt/optA) Or CTG (optB).This identical process is repeated for all leucines in this sequence, and due to the random nature of method, CTC Codon may terminate at optB and CTG and may terminate at optA.Due to the optimization of this two codons using method, sequence opt/optA The codon maximum with opt B is different.
Table 17: codon used in codon optimized
Aminoacid Select #1 Select #2
Gly GGC GGG
Glu GAG GAA
Asp GAC GAT
Val GTG GTC
Ala GCC GCT
Ser AGC TTC
Lys AAG AAA
Asn AAC AAT
Met ATG
Ile ATC ATT
Thr ACC ACA
Trp TGG
Cys TGC TGT
Tyr TAC TAT
Phe TTC TTT
Arg CGG CGC
Gln CAG CAA
His CAC CAT
Leu CTG CTC
Pro CCC CCT
4 sequences (IV opt, IV optB analysed as described above；9B8opt, 9B8optB) in each transcribe because of The existence (Genomatix Software, Germany) of sub-binding site or other regulating and controlling sequences, and changed by silent nucleotide Become and destroy these unwanted sequences.In some cases, wherein there are the mankind and other non-rare codons colibacillary Time, remove Binding site for transcription factor or other controlling elements, even if they are not by changing into one of these codons Select #1 or select #2 (seeing table 18).In such cases, Binding site for transcription factor or other controlling elements are wherein removed To relate to importing rare codon, the most do not change transcript binding site (or other controlling elements).
Table 18: for removing the other codon of Binding site for transcription factor and other controlling elements
Aminoacid Select #3 Select #4
Gly GGA GGT
Val GTA GTT
Ala GCG GCA
Ser AGT TCA
Thr ACG ACT
Leu TTG CTT
Pro CCG CCA
IV (" IV opt " (SEQ ID NO:22) and " IV optB " (SEQ ID is generated by methods known in the art ) and the codon optimized shape of 9B8 (" 9B8opt " (SEQ ID NO:24) and " 9B8optB " (SEQ ID NO:25)) NO:23) Formula is also cloned in pF4Ag.HEK293 cell is seeded in 96 hole flat boards and at 37 DEG C with 15,000 cells/well Overnight growth.Second day, utilize the plasmid DNA encoding codon optimized form in pF4Ag of 100ng, utilizeTransfection Reagent (Mirus Bio) by cell transient transfection to 6 holes repeat in and Overnight incubation at 37 DEG C.Also utilize pGL4.13 (Luc2/SV40) (Paguio etc., " pGL4Vectors:A New Generation of Luciferase Reporter Vectors " Promega Notes, 89:7-10 (2005)) or PGL4.73 (hRL/SV40) (ditto) has transfected HEK293 cell and has been standardized with the difference for transfection efficiency.Employ 10ng/ transfection or the STb gene transfection of 10%.Remove culture medium, and utilize 100 μ L lysis buffer cell lysis to generate cracking Sample, described lysis buffer contains 10mM CDTA, 150mM KCl, 10mM DTT, 100mM HEPES, pH7.0,35mM sulfur Urea, and 0.5%NP-9(v/v).Exist as followsMeasure on F500 photometer The luminescence of lysate sample: for hRL and OgLuc variant, utilizes the 50 μ L lysis buffers containing 20 μMs of substrates to measure cracking sample The luminescence of product (be natural coelenterazine for hRL and be PBI-3939 for OgLuc variant).For Luc2 (SEQ ID NO:28 With 29), LUC Photinus pyralis LUC Photinus pyralis FL, utilize the BRIGHT-GLOTM Luciferase of 50 μ L to measure reagent (Promega Corp.) Measure the luminescence of 10 μ L lysate sample.
Figure 32 show luminescence and hRL that the lysate for the codon optimized form containing OgLuc variant measures and The comparison of Luc2.Utilize this area known method by hRL and OgLuc variant about pGL4.13 standardization and by Luc2 close In pGL4.73 standardization.As shown in Figure 32, Luc2 has the luminescence of high about 14 times than hRL.OgLuc variant and Luc2 and hRL Compare and there is higher luminescence.IV (" IV opt " and " IV optB ") and the codon optimized form of 9B8 (" 9B8opt ") Show the luminescence increased compared with unoptimizable form.
As the result optimized, " opt/optA " form is expressed more than its parental sequences in mankind's HEK293 cell Good, and " optB " form does not has same expression in HEK293 cell compared with parental sequences.
B.L27V
Optimize L27V variant (SEQ ID NO:88) with by common vertebrates response element (in Genomatix data base Any Binding site for transcription factor (TFBS)) minimize.Construct the shape of three kinds of different optimizations of L27V variant Formula:
1.L27V01-form 1 (SEQ ID NO:319)-and in addition to single TFBS, eliminate startup by nucleotide subsitution Sub-component and all other unwanted sequential element (other further below literary compositions).
2.L27V02-form 2-L27V01 is used as homing sequence, i.e. parental sequences, and utilizes high stringency to mate standard (higher stringency relates to mating with the more preferable of binding site and therefore sending out than relatively low stringency removal TFBS as far as possible The most less coupling).Construct two kinds of forms of L27V02, A (SEQ ID NO:322) &B ((SEQ ID NO:318)).These are two years old Kind of form is by building to remove unwanted sequential element the codon that every kind of formal character is different.Relatively low by utilizing Stringency scans for analyzing two kinds of forms about TFBS.
3.L27V03-form 3 (SEQ ID NO:325)-L27V02B (SEQ ID NO:318) is used as homing sequence.Can The TFBS coupling of relatively low stringency is removed during energy.Building L27V03 is the codon very different with L27V02A.
Employ following standard to build the variant that L27V optimizes:
1. codon uses: preferably, uses two kinds of optimal human codon (as about IV variant in every kind of aminoacid Done), and avoid one among a thousand class codon (HS；Coding ＜ 10% aminoacid) use (table 19).If desired, use Rare e. coli codon (EC) is to remove unwanted sequential element.
Table 19
2. when possible, remove unwanted sequential element
A. enzyme (RE) site is limited: remove and to cloning useful RE site and or may should be not present in open reading code In frame (ORF).
B. eucaryon sequential element: remove (+) donor splicing site in mRNA chain and acceptor site, montage silencer, Kozak sequence Row and polyadenylic acid sequence.
C. vertebrate promoter assembly (PM) (in Genomatix catalogue: vertebrates) is removed.
D. may time remove vertebrates TFBS (in Genomatix catalogue: vertebrates, general core promoter Element and other sequences various).This is only applied to L27V and optimizes form 2 and 3, and shall not be applied to form 1.
E. escherichia coli sequential element: eliminate escherichia coli promoter.
F.mRNA secondary structure: remove nearly 5 ' the strong secondary structures held (high mRNA folds energy) (Zuker, Nucleic Acid Res.31 (13): 3406-3415 (2003)) and other strong hairpin structures.
Table 20 provides sequence alignment, paired Percentage of sequence identity (number of " () " expression nucleotide difference).
Table 20
L27V01 L27V02A L27V02B L27V03
L27V00 99% (3) 97% 97% 94%
L27V01 98% (12) 98% 94% (32)
L27V02A 99% (4) 95% (26)
L27V02B 96%
The signal stabilization of embodiment 26 OgLuc variant
A.15C1,9B and IV
The signal stabilization utilizing PBI-3945 to measure 15C1 and the signal stabilization of the 9B8 utilizing PBI-3889 to measure And compared with IV.Cultivate the escherichia coli containing the plasmid DNA encoding 15C1,9B8 or IV, and as described earlier in 8 holes Duplication is induced.Utilize the HEPES pH8.0 Han 300mM, the passive lysis buffer of 0.3X (" PLB "；Promega Corp. catalogue Number E194A) 0.3mg/mL lysozyme and the lysis buffer cell lysis of 0.003U/ μ L RQ1DNA enzyme.By lysate with 1: 1000 are diluted in lysis buffer and utilizeF500 photometer measurement is luminous.To 10 μ L Dilution lysate sample in add the KCl Han 150mM, 1mM CDTA, 10mM DTT, 100mM thiourea, 0.5%" Glo " 0.5%TERGITOL of the 50 μ L of NP-9 (v/v) measure buffer (" 0.5%TERGITOL ") and Measure immediately after novel coelenterazine PBI-3945 of 20 μMs or PBI3889.
Re-read flat board by a period of time after adding mensuration buffer to sample every 30 seconds and determine variant Signal stabilization.Method as known in the art is utilized to determine the signal half-life from these measurement results.At variant and IV Between compare the average signal half-life.15C1 and 9B8 has the signal half-life (Figure 33) of at least 30 minutes.Although utilizing PBI- 3945 15C1 measured have a higher luminescence when t=0, but signal attenuation obtains variant that Billy measures with PBI-3889 more Hurry up.When t=10 minute, identical when utilizing luminescence and the 9B8 utilizing PBI-3889 luminous of the 15C1 of PBI-3945.
B.9B8opt+K33N
Checked the signal stabilization of 9B8opt+K33N variant.It is prepared for the E. coli lysate containing variant and as first Being analyzed described by before, except for the difference that measures buffer containing 0.25%NP-9 (v/v), 100mM MES PH6.0,1mM CDTA, 150mM KCl, 35mM thiourea, 2mM DTT, and 20 μMs of PBI-3939.Table 22 show variant with The signal half-life of minute meter, and show that amino acid replacement L27V improves signal stabilization.
Table 22:OgLuc variant signal stabilization in bacterial lysate
Measure L27V variant (9B8+K33N+L27V+T39T+K43R+Y68D；SEQ ID NO:88 and 89) signal activity Compared with belonging to luciferase with stability and with LUC Photinus pyralis LUC Photinus pyralis FL and sea pansy.By L27V variant, Luc2 luciferase and Sea pansy belong to luciferase withMelt and be incorporated in expression in escherichia coli.UtilizeAs purification Label is according to the scheme purification luciferase (pFN18A of manufacturer；Protein Purification System).By the luciferase of every kind of purification of 10pM (without phenol red containing 0.01%DMEM in dilute Release) and isopyknic mensuration reagent (it is 100mM MES pH6 for L27V variant, 35mM thiourea, 0.5% NP-9 (v/v), 1mM CDTA, 2mM DTT, 150mM KCl, and 100 μMs of PBI-3939；For LUC Photinus pyralis LUC Photinus pyralis FL it is ONE-GLOTMLuciferase Assay System(Promega Corp.)；It is RENILLA-with belonging to luciferase for sea pansy GLOTMLuciferase Assay System (Promega Corp.)) mix mutually, and passage (3,10,20,30,45 in time With 60 minutes) monitoring luminescence.Figure 34 A-B indicates L27V time compared with belonging to luciferase with LUC Photinus pyralis LUC Photinus pyralis FL and sea pansy The high specific acitivity (Figure 34 A) of variant and signal stabilization (Figure 34 B).
The enzyme kinetics of embodiment 27 OgLuc variant
A.IV, 15C1,9B8,9F6 and 9A3
Utilize method as known in the art, utilize and contain the colibacillary of IV and IV variant 15C1,9B8,9F6 and 9A3 Lysate measures the enzyme kinetics of luminescence and measures.Induction, cracking and diluting cells as described in embodiment 26, different Be lysis buffer pH be 7.5.Lysate as previously utilizing described in embodiment 26 diluted measures buffering The PBI-3939 of the twice serial dilution in liquid.Figure 35 shows and utilizes IV and the hyperbola of variant 15C1,9B8,9F6 and 9A3 Km and the Vmax value of the Fitting Calculation.Variant 9B8 with 9F6 has higher Km compared with IV, and the Km of other variants is constant.With IV compares, and variant 15C1,9B8 and 9F6 are respectively provided with higher Vmax value, and 8A3 has relatively low Vmax value.
15C1, it utilizes PBI-3945 to have the highest luminescence, containing amino acid replacement K33N, shows that K33N provides and increases Luminescence.Generate 9B8 variant so that this is additionally replaced into the increase that this variant provides luminous.Generate 9B8 and 9F6 additionally Variant to have at least one (" 9B8+K33N+V38I " and " 9F6+K33N ") in amino acid replacement K33N or V38I.Become Body 1D6 is for strengthening the amino acid replacement of the 68th, 72 and 75 for increasing light output and the importance of stability.Figure 36 shows Utilize Km that the hyperbolic fit about IV and variant 9B8,9B8+K33N+V38I, 9F6,9F6+K33N and 1D6 calculates and Vmax value.Although be different about the actual Km value between Figure 35 and 36 of 9B8 and 9F6, but the general trend between variant It is consistent.
Determine enzyme kinetics, i.e. Vmax and Km value is also carried out for variant 9B8opt and 9B8opt+K33N as previously mentioned Relatively, except for the difference that utilization CTDA Han 1mM, 150mM KCl, 2mM DTT, 100mM MES pH6.0,35mM thiourea, 0.25%NP-9 (v/v), the buffer of 10mg/mL2-HP-β-CD and 20 μMs of PBI-3939 measures big Enterobacteria lysate.?Luminescence is measured on F500 photometer.As shown in Figure 37,9B8opt Vmax and the Km value of+K33N is higher than 9B8opt, shows that this clone is brighter and have have relatively low affinity to substrate.
B.9B8OPT+K33N variant
Determine the enzyme kinetics value of OgLuc variant as described earlier, except for the difference that utilizeLuminosity Measurement amount is luminous.Three repetitions are used for each variant.Table 23 shows and utilizes HYPER.EXE, and what version 1.0 calculated has Average Km and Vmax of standard deviation is (respectively " Km (+/-) " and " Vmax (+/-) ".
The Vmax value (RLU/0.5 second) of table 23:OgLuc variant and Km (μM) value
The protein stability of embodiment 28 OgLuc variant
Owing to the stability of luciferase protein is impact another factor luminous, it is determined that protein stability, i.e. thermally-stabilised Property.
A.15C1,9B8,9F6,8A3 and IV
Be prepared for from the cultivation of induction as described earlier the escherichia coli containing 15C1,9B8,9F6,8A3 or IV and Express the colibacillary lysate of hRL (SEQ ID NO:30 and 31).With the 10mM HEPES comprised containing 0.1% gelatin The buffer 1: 1000 of pH7.5 dilutes lysate sample.Splitting of the dilution in two 96 hole flat boards repeated is hatched at 50 DEG C Solve thing (100 μ L) sample.At different time points, flat board is placed in-70 DEG C (subzero 70 degrees Celsius).As described earlier Before measuring luminescence, at room temperature that is 22 DEG C, melt each flat board 10 minutes.Natural coelenterazine is utilized to measure sample as substrate (sample of each fusion of 10 μ L).After adding mensuration buffer, luminescence is measured immediately for each time point flat board.Profit By method as known in the art, each time point is calculated from light-emitting data the half-life of the albumen of indicator protein stability.
Table 24 show be respectively provided with minute (hour) count for 630.1 (10.5), 346.6 (5.8), 770.2 (12.8) protein stability of variant 15C1,9B8,9F6 and 8A3 of and 65.4 (1.1).By comparison, hRL has 9.6 minutes Half-life, and IV has the half-life of 27.2 minutes.Table 24 also show 4 little constantly, 15C1,9B8, and the difference of 9F6 It is 79%, 61%, and 80%, keep activity.
The protein stability of OgLuc variant during 24:50 DEG C of table
Sample Half-life (minute) Half-life (hour) Little residue % constantly of t=4
Sea pansy belongs to 9.6
IV 27.2
15C1 630.1 10.5 79%
9B8 346.6 5.8 61%
9F6 770.2 12.8 80%
8A3 65.4 1.1
B.1D6,9B8,9B8+K33N+V38I, 9F6,9F6+K33N, and IV
The colibacillary of 1D6,9B8,9B8+K33N+V38I, 9F6,9F6+K33N or IV is contained from the cultivation preparation of induction Lysate, and measure luminescence as described earlier.Protein stability the most determined as outlined above, i.e. lysate Heat stability.Figure 38 shows the variant half-life at 50 DEG C in minutes, and utilizes natural coelenterazine to exist as substrate Hatch the luminescence of the initial i.e. sample that t=0 measures of period.Difference between variant 9B8+33+38 and 9F6 is an aminoacid Displacement, L27V, show that this amino acid replacement increases stability.The increase that " activity/express " of the 68th, 72 and 75 is replaced carries High stability.Figure 38 shows that provided K33N is that variant 9F6 provides higher heat stability and variant 9B8 compares variant 1D6 has the output of higher light and stability.Difference between these two kinds of variants, i.e. 9B8 is containing other amino acid replacement F68Y, L72Q, and M75K, indicate the importance of these three kinds displacements.
Outside heat extraction stability, expression structural intergrity, stability and the dissolubility determined also can affect luminescence.As entering The mode of the structural intergrity of the variant of one step test improvement, will contain OgLuc variant N166R based on pF4Ag (that is, without HT7) (being previously described in U.S.Application Serial the 12/773,002nd (U.S. Published Application the 2010/0281552nd)), C1+A4E, The KRX escherichia coli of IV, 9B8 and 9F6 are incubated at Luria meat soup (LB) to OD at 37 DEG C600=0.6 and then by add Rhamnose (0.2% final concentration) induction process LAN.Then at 25 DEG C or 37 DEG C, the cultivation 17 hours repeating induction is cultivated, at this Time prepares total (T) fraction and dissolves (S) fraction and utilize SIMPLYBLUE by SDS-PAGETMSafeStain (Invitrogen) (Figure 39 A-B) it is analyzed with stained gel.HRL and Luc2 is with comparing.
When induction occurs at 25 DEG C, OgLuc variant, hRL and Luc2 good representation and be solvable (Figure 39 A；Note For the about 19kDa black stripe in OgLuc variant " solvable " fraction, remove N166R variant, for hRL and Luc2 " solvable " The most about 36kDa band and 64kDa band in fraction).By contrast, although C1+A4E, IV, 9B8 and 9F6 express at 37 DEG C Well (as shown in " always " fraction, be significantly better than hRL or Luc2), when using the inducing temperature increased, only 9B8 and 9F6 Variant is solvable (to see Figure 39 B；Note for 9B8 and 9F6 about 19kDa black stripe in " solvable " part).These knots Fruit is consistent with the thermal stability data shown in table 24 and Figure 38.
And 9B8OPT+K33N C.9B8OPT
Compare the heat stability of variant variant 9B8opt and 9B8opt+K33N.Be prepared for containing variant 9B8opt or The E. coli lysate of 9B8opt+K33N is also analyzed the most as described earlier: previously describe In lysis buffer, dilute lysate and in thermocycler 60 DEG C at hatch the lysate that repeat dilution at 1: 100.Not Same time point is removed equal portions and is placed on dry ice with freezing sample.At 22 DEG C, lysate the utilization of melting chilling contains 20mM CDTA, 150mM KCl, 10mM DTT, 20 μMs of PBI-3939,100mM HEPES pH7.0,35mM thiourea, and 0.1%The buffer of NP-9 (v/v) is measured.?On photometer (Promega Corp.) Measure luminescence.Figure 40 A shows the light elapsing the luminous natural logrithm value that (in minutes) measures in units of RLU in time Output time process.As shown in Figure 40 B, 9B8opt+K33N has the half-life of 6.8 hours at 60 DEG C, its ratio The half-life of 5.7 hours of 9B8opt to be grown.
Table 25 shows 9B8opt and 9B8opt+K33N heat stability (" T at 60 DEG C1/2(60 DEG C) "), and hatching Luminescence (" RLU ") data during initial (that is, the t=0) of time.9B8opt+K33N is more stable and brightness ratio 9B8opt is high by about 1.8 Times, show that amino acid replacement K33N provides the output of higher light and higher heat stability.
The heat stability of table 25:9B8opt and 9B8opt+K33N and light-emitting data
D.9B8+K33N variant
The heat stability of the variant at checking 60 DEG C as previously mentioned, except for the difference that measures buffer MES Han 100mM PH6.0 rather than HEPES.Table 26 and Figure 41 shows the half-life in hours of variant at 60 DEG C.Data show aminoacid Displacement L27V improves heat stability.
Table 26:OgLuc variant heat stability at 60 DEG C
In HEK293 cell, also screen variant 9B8 and V2 (9B8+K33N+T39T+K43R+Y68D) to determine that it is steady Qualitative.As described earlier by variant clone to pF4Ag and be transfected in HEK293 cell (15,000 cells/well). After transfection, in measuring reagent, cell lysis is (as described earlier；Without PBI-3939), utilize containing 20 μMs of PBI-3939's Mensuration reagent measuring is luminous.9B8 has the half-life of 5.2 hours, and V2 has the half-life of 16.8 hours.This with at large intestine bar The half-life found about these variants in bacterium is consistent (table 26).
E.L27V variant
L27V variant (9B8+K33N+L27V+T39T+K43R+ is evaluated with under the conditions of different salt under different pH Y68D) activity.Previously showed that 9B8 with 9B8+K33N had similar stability (data are not shown) under pH6 with pH7. For evaluating the activity under the conditions of different salt, the 50 μ L containing 20 μMs of PBI-3939 are measured buffer and the KCl of different amounts Or the HEK293 cell of NaCl and the 50 μ L utilizing L27V (pF4Ag) transient transfection mixes mutually.Measure luminescence, determine activity percentage Than (luminous and salt-free ratio) (Figure 42 B).For evaluating the activity in different pH, it is prepared for containing 100mM citrate, 100mM MES, 100mM PIPES, 100mM HEPES, 100mM TAPS, 0.5%NP-9 (v/v), 0.05%DF204,1mM CDTA, and the reagent of 1mM DTT, for different pH value titration.To measure in reagent 100 μMs of PBI-3939 phases of 362pM L27V and substrate mix and measure luminescence (Figure 42 A).
The analysed by gel filtration chromatography of embodiment 29 OgLuc variant
A.C1+A4E and 9B8
Gel-filtration analysis is used to verify the desired molecular weight of OgLuc albumen of purification based on theoretical value, and thus Determine its oligomeric state.By gel filtration chromatography carry out OgLuc variant C1+A4E and 9B8 fluid relative hydrodynamic volume it Between comparison.For carrying out this analysis, the nucleotide sequence of OgLuc variant C1+A4E and 9B8 is cloned into HQ-TaggedTo build the HQHQHQ N-end of process LAN in escherichia coli KRX cell in Vector (Promega Corp.) The albumen of labelling.Utilize HISLINKTMProtein Purification System (Promega Corp.) is according to manufacturer The albumen of purification process LAN is described.By each single standard of analysed by gel filtration chromatography and the sample of sample protein, 24 Carry out on Agilent1200HPLC at DEG C, utilize the Superdex2005/150GL post (GE that flow velocity is 0.25mL/ minute Healthcare).Flowing phase (that is, electrophoretic buffer) is made up of 50mM Tris and 150mM NaCl, pH7.5.214 Hes Albumen eluting is monitored at 280nm.Standard calibration curve is generated: 1 below utilizing) ovalbumin, 43kDa (GE Healthcare), 2) carbonic anhydrase, 29kDa (Sigma) and 3) Myoglobin, 17kDa (Horse Heart, Sigma).Directly The molecular weight of the albumen of purification is calculated from calibration curve.
The relative elutropic utilizing the albumen that this post observes when 7.98 minutes is ovalbumin, is carbonic acid when 8.65 minutes Acid anhydride enzyme, was 9B8 when 8.97 minutes, and was Myoglobin (Figure 43 A-B) when 9.06 minutes.As shown in Figure 43 B, 9B8 washes Take off for 21kDa albumen (molecular weight of prediction is about 19kDa), show that 9B8 exists as monomer, and C1+A4E is at about 4.3 points Eluting (Figure 43 A) during clock, shows that C1+A4E is expressed and exists as polymer, for example, it may be possible to as tetramer complex Or bigger complex.
B.L27V variant
For proving that OgLuc variant L27V exists with free state, gel-filtration analysis is utilized to verify based on theoretical value pure The desired molecular weight of the L27V albumen changed, and so that it is determined that its oligomeric state.The phase of L27V is carried out by gel filtration chromatography Convection cell hydrodynamic volume.For carrying out this analysis, the nucleotide sequence of L27V variant is cloned intoVector To build process LAN in escherichia coli KRX cell in pFN18A (Promega Corp.)End-labelled Albumen.UtilizeProtein Purification System (Promega Corp.) is according to the explanation of manufacturer The albumen of purification process LAN.By each single standard of analysed by gel filtration chromatography and the sample of sample protein, at 24 DEG C Agilent1200HPLC is carried out, utilizes the Superdex2005/150GL post (GE that flow velocity is 0.25mL/ minute Healthcare) (Figure 56).Flowing phase (that is, electrophoretic buffer) is made up of 50mM Tris and 150mM NaCl, pH7.5.? Albumen eluting is monitored at 214 and 280nm.Standard calibration curve is generated: 1 below utilizing) ovalbumin, 43kDa (GE Healthcare), 2) Myoglobin, 17kDa (Horse Heart, Sigma) and 3) ribonuclease, 14kDa (Bovine Pancreas, GE Healthcare).As shown in Figure 44, L27V variant eluting is that (predicted molecular weight is about 24kDa albumen 19kDa), show that it exists as monomer.
The protein expression level of embodiment 30 OgLuc variant
A.IV, 8A3,8F2,9B8,9F6 and 15C1
The standardization of protein expression provides the information of the possible difference about specific activity.Protein expression is quantified for providing Mode, as described earlier by OgLuc variant clone to the pF4Ag carrier containing C-end HT7 with generate OgLuc variant- HT7 fusion protein.Generate following fusion protein: IV-HT7 (SEQ ID NO:48 and 49), 8A3-HT7 (SEQ ID NO: 34 and 35), 8F2-HT7 (SEQ ID NO:50 and 51), 9B8-HT7 (SEQ ID NO:36 and 37), 9F6-HT7 (SEQ ID NO:38 and 39), and 15C1-HT7 (SEQ ID NO:52 and 53).Make the Escherichia coli Growth containing OgLuc variant-HT7 fusant And induce as described earlier.10X FASTBREAK with 100 μ LTMCell lytic reagent (Promega Corp.) splits Solve the cell culture of 900 μ L.WillTMR-part (Promega Corp.) adds each bacteria lysis to Thing sample is with the final concentration obtaining 0.5 μM.At room temperature utilize according to the explanation of manufacturerFMR-part is incubated Educate bacterial lysate 30 minutes.Utilize 1X FASTBREAKTMDilute every kind of sample of 10 μ L, i.e. add 10 μ L sample to 10 μ L1X FASTBREAKTM.Every kind of sample is analyzed to lysate and 1: 1 diluent of 15 μ L of 15 μ L by SDS PAGE.Pass through SDS-PAGE resolves the fusion protein of labelling, uses SIMPLYBLUETMSafeStain (Figure 45 A) and phosphorimager (GE Healthcare Typhoon) dyeing.Utilize Imagequant software (GE Healthcare) quantitatively band.Figure 45 B shows From about IV-HT7 (" IV "), 15C1-HT7 (" 15C1 "), 9B8-HT7 (" 9B8 "), 9F6-HT7 (" 9F6 "), and 8F2-HT7 The band volume that Figure 45 A of (" 8F2 ") measures, about IV-HT7 standardization.Data show that IV variant is good compared with IV Express.
B.9B8opt, V2 and L27V
Compare expression and the dissolubility of 9B8opt, V2 and L27V.In pF4Ag background environment, use these three kinds Variant converts escherichia coli KRX cell.The clone of gained is used for expressing experiment, wherein grows overnight with 1: 100 at 30 DEG C The single clone being diluted in LB, grows to OD600About 0.5, and then induce 18 hours with 0.2% rhamnose at 25 DEG C. Then at 0.5X FASTBREAKTMAt room temperature incubated cell 30 minutes in the presence of lytic reagent (Promega Corp.), and will At gained lysate is stored in-20 DEG C.After slowly melting on ice, within 10 minutes, prepare solvable by high speed centrifugation at 4 DEG C Fraction.Then utilize SDS-PAGE+ simply blue dyeing (Figure 46 A) and by measure thick total (T) fraction of luminesceence analysis and The expression of solvable (S) fraction.For carrying out luminous measurement, 50 lysates solvable for μ L in 96 hole microtiter plate and 50 μ L are measured examination Agent is (previously described；40 μ L PBI-3939) mix mutually, and utilizeThe many detections of F500 are put down Plate reader measures luminescence.These results show that these three kinds of variants are ordered as L27V ＞ V2 about its expression and dissolubility ＞ 9B8opt.
The brightness of the OgLuc variant that embodiment 31 is expressed in mammalian cell
A.IV and 9B8
The brightness of IV and the 9B8 variant in assessment pF4Ag carrier (that is, without HT7) in HEK293 cell.It is right that hRL is used as According to.In short, will be with 15, the HEK293 cell that 000 cells/well is seeded in 96 hole flat boards utilizes the variant different containing coding The plasmid DNA of sequence and/or control sequenceCarry out transient transfection.As described in embodiment 25 Cultivate cell, crack and process.PGL4.13 (Promega Corp.) is utilized (to use as transfection control cotransfection cell The 10% of the STb gene of 10ng/ transfection or transfection).Natural coelenterazine is utilized as the substrate of hRL or to utilize PBI-3939 conduct The substrate of OgLuc variant measures luminescence as described earlier.Utilize Luc2 luminous (that is, at interpolation fluorescence about transfection efficiency Luminescence is measured after element substrate) correction OgLuc variant data.OgLuc variant IV and 9B8 has higher luminescence compared with hRL (Figure 47).
For carrying out the comparison of brightness in mammalian cell on every mole foundation, to the change described in embodiment 30 The HT7 fusion protein (" pF4Ag-OgLuc-9B8-HT7 ") of body 9B8 be analyzed and with C-end HT7-hRL fusion protein (" pF4Ag-sea pansy genus-HT7 ") compares with C-end HT7-Luc2 fusion protein (" pF4Ag-Luc2-HT7 ").Inoculation HEK293 cell (15,000) also grows overnight at 37 DEG C.Utilize from pF4Ag-Renilla-HT7, pF4Ag-Luc2-HT7 Or the DNA of the 100ng of pF4Ag-OgLuc-9B8-HT7 transfects these cells and grows overnight at 37 DEG C.Remove culture medium also Cell lysis as described earlier.Every kind of sample determination luminescence (RLU) to 10 μ L, utilizes the 50 μ L for Luc2 BRIGHT-GLOTMLuc2, for 20 μMs of natural coelenterazine of the 50 μ L of hRL, and 20 μMs of PBI-of the 50 μ L for variant 9B8 3939。
Mix from the lysate in 6 holes the use as described in embodiment 30TMR-part is carried out Labelling.Resolve the fusion protein of labelling by SDS-PAGE and carry out fluorescence imaging (GE Healthcare Typhoon).Determine Ribbon density is with the relative molar amounts quantitatively existed about every kind of luciferase, and carrys out standardization every kind by calculating band concentration The expression of albumen and standardization are about the RLU value of every kind of sample, i.e. utilize TMR labelling quantitatively to carry out standardization RLU (figure 48).On mol ratio mole foundation, 9B8 variant is higher about 15 times than Luc2 brightness, and higher about 100 times than hRL.These data represent The difference of specific activity.
And 9B8opt+K33N B.9B8opt
Measure the brightness of variant 9B8opt and 9B8opt+K33N expressed in HEK293 cell, and as in embodiment 31 About without comparing described by the variant of HT7.The plasmid DNA using containing modification D NA 30 and 100ng transfects HEK293 cell.Growth cell the carrying out as described in embodiment 31 are induced, and except for the difference that utilize the CTDA Han 1mM, 150mM KCl, 2mM DTT, 100mM MES pH6.0,35mM thiourea, 0.25%NP-9 (v/v), and The lysis buffer cell lysis of 10mg/mL2-HP-β-CD.Survey with the lysis buffer containing 20 μMs of PBI-3939 Determine lysate andGENIOSTMLuminescence is measured on Pro photometer.As shown in Figure 49, in HEK293 cell 9B8opt+K33N has higher luminescence compared with 9B8, and this is consistent with the bacterial expression data in table 25 and Figure 29.
V.9B8+K33N variant
The brightness measuring the variant expressed in HEK293 cell and NIH3T3 cell as described earlier.Sending out variant The emission standards (table 27) that light produces about 9B8opt.
The luminous increase produced by OgLuc combinatory variants in table 27:NIH3T3 cell and HEK293 cell
D.L27V
Carry out L27V variant and single LUC Photinus pyralis LUC Photinus pyralis FL and the luminescence of the LUC Photinus pyralis LUC Photinus pyralis FL as fusant Comparison.With 15,000 and 10,000 cells/well, HEK293 cell and HeLa cell are inoculated into the hole of 12 hole flat boards respectively In and at 37 DEG C, 5%CO2Lower night incubation.Then the pF4Ag transfectional cell containing L27V or Luc2 of serial dilution is utilized. The pGL4.13 (Promega Corp.) of 20ng and L27V cotransfection, and the pGL4.73 (Promega Corp.) and Luc2 of 20ng Cotransfection is for use as the carrier DNA for L27V or Luc2 plasmid DNA.Then utilizeTransfection reagent root According to the explanation of manufacturer by plasmid DNA transfection to cell (for 6 repetitions of every kind of each dilution of cell type).Then 37 At DEG C, 5%CO2Lower incubated cell 24 hours.
After transfection, remove culture medium from cell, and add containing 0.5%The 100 μ LPBS of NP-9 (v/v) And at room temperature vibrate 10 minutes.Utilize ONE-GLOTMLuciferase Assay System(Promega Corp.；Luc2) Or (embodiment 22H contains 20 μMs of PBI-3939 to measure reagent；OgLuc).As described earlier about HEK293 cell (figure 50A) measure luminescence with HeLa cell (Figure 50 B).
The comparison of L27V and Luc2 as fusion partner proceeded as above.By in L27V and Luc2 and pF4AgProtein fusion.Figure 50 C-D shows and utilizes in HEK293 cell (Figure 50 C) and HeLa cell (Figure 50 D) The luminescence that different fusants is measured.
In addition to measuring luminescence, also analyze protein expression.Transfection proceeded as above.After transfection, remove from cell and cultivate Base, and washed cell in 1X PBS.Add containing 1 μMPart (Promega Corp.) and 20U 100 μ L0.1X mammal lysis buffer (Promega Corp.) of DNA enzymatic I, and at room temperature hatch under slowly vibration Cell 45 minutes.Then frozen cell sample at-20 DEG C.For analysis of protein, 32.5 μ L4X SDS loading dye are added To every kind of sample, and sample is heated 2 minutes at 95 DEG C.Then 10 μ L sample are loaded on PAGE gel, and such as Previously described imaging on Typhoon Scanner (Figure 50 E).
Embodiment 32 is the brightness of the OgLuc variant of purification compared with LUC Photinus pyralis LUC Photinus pyralis FL
Such as the process LAN described in embodiment 33 purification 9B8OgLuc variant.Utilize following 2X buffer/survey Determine reagent to react between enzyme and the substrate of dilution: 100mM MES pH6.0,1mM CDTA, 150mM KCl, 35mM sulfur Urea, 2mM DTT, 0.25%NP-9 (v/v), 0.025%DF204,10mg/mL2-hydroxyl- Beta-schardinger dextrin-, and 20 μMs of PBI-3939.The enzyme of purification and substrate final measures concentration and is respectively 0.5pM and 10 μMs.Parallel Ground, analyzes the LUC Photinus pyralis LUC Photinus pyralis FL of purification of dilution (i.e.,Recombinant Luciferase (Promega Corp.)) and fluorescein between reaction.Mensuration buffer/the reagent of LUC Photinus pyralis LUC Photinus pyralis FL reaction is BRIGHT-GLOTM, and finally mensuration concentration is 0.5pM enzyme and 500 μMs of fluoresceins.Due to buffer known for each reaction/ Reagent provides " aura " kinetics, uses 15 minutes points to gather light-emitting data.Result from this experiment shows utilization The 9B8opt brightness ratio of PBI-3939 (19,200RLU) utilizes BRIGHT-GLOTM(2,300RLU) Recombinant Luciferase is high about 8 times.
Embodiment 33 inhibition analysis
For determining the OgLuc variant sensitivity for interaction of missing the target, for the LOPAC (literary composition of pharmaceutically active compounds Storehouse) library screening 9B8 and the activity of L27V variant.LOPAC1280 literary composition is prepared by compound is diluted to 1mM in DMSO Storehouse (Sigma).Measuring the same day, compound is being diluted to 20 μMs in 1X PBS, and 10 μ L are transferred to 96 hole white plate. It is added in Glo Lysis Buffer (Promega Corp.) to every hole with 10-4The 9B8, L27V of the purification of 10 μ L of dilution Or LUC Photinus pyralis LUC Photinus pyralis FL (Lu2) at room temperature hatch 2 minutes.Add in sample 20 μ L measure reagent (1mM CDTA, 150mM KCl, 2mM DTT, 100mM MES pH6.0,35mM thiourea, 0.5%NP-9 (v/v) and 60 μMs PBI-3939), hatch 3 minutes, andGENIOSTMLuminescence is measured on Pro photometer.For measuring Lampyridea fluorescence Element enzyme, uses BRIGHT-GLO according to the scheme of manufacturerTMMeasure reagent (Promega Corp.).As negative control, each 8 holes PBS+2% glycerol Han 1X of flat board.As positive control, 8 holes of each flat board are contained in the 2mM in 2%DMSO Suramin or the 2mM luciferase inhibitor 1 (Calbiochem) being contained in 2%DMSO.In the primary screener in LOPAC library (that is, utilizing the 9B8 variant screening LOPAC library of the relatively low concentration of substrate with 20 μMs) identifies the suppression that suramin is OgLuc Agent.
Result in Figure 51 shows the general low frequency interacted that misses the target between the compound in LOPAC library and L27V Rate.This shows that L27V is as different chemicals and the large-scale library for the treatment of material standed for for including form based on living cells The potential use of the screening implement of (such as, high flux screening).
For checking inhibition further, for suramin (Sigma S-2671) and the Tyrphostin of variable concentrations 9B8 and L27V (Figure 52 A-C) of AG835 (" Tyr ag835 ") (Sigma T-5568) screening purification.Figure 52 E-D shows respectively The chemical constitution of Suramin and Tyr ag835.9B8 and L27V of purification prepared as described above.At the 1X containing 2%DMSO PBS is prepared the serial dilution (0,2 μM, 6 μMs, 20 μMs, 60 μMs, 200 μMs and 2mM) of inhibitor.To 96 hole white assay plates Hole in add the enzyme of dilution of 10 μ L and the inhibitor of the dilution of 10 μ L and at room temperature hatch 2 minutes.Add 20 μ L to measure Reagent (as mentioned above), andMeasure luminous (Figure 52 A-C) on photometer.Figure 52 A-B shows 9B8 With L27V for suramin (Figure 52 A) and the dose response curve of Tyr ag835 (Figure 52 B).Figure 52 C show for 9B8 and The half maximum inhibition concentration (IC of L27V suramin and Tyr ag83550).Data show that L27V can be used as different chemistry systems The firm reporter of the screening implement in the large-scale library of product and/or treatment material standed for.
The resistance that nonspecific proteins is interacted by embodiment 34
1. by 9B8 and the L27V enzyme of purification with or without 0.5mg/mL BSA buffer (1X PBS, 1mM DTT and 0.005%CA-630) it is diluted to 200 μ L in PCR pipe with 1: 10 in.Sample, Qi Zhong is hatched at 60 DEG C One group of diluent of each variant little is transferred to-70 DEG C by 0,2,4 and 6 constantly.
For analyzing activity, in a water bath sample is melted to room temperature.The mensuration reagent adding 50 μ L is (as described earlier Containing 100 μMs of PBI-3939), andOn F500 flat bed reader, measurement per minute is sent out Light continues 30 minutes.Utilize 1x106And 1x107The average luminescence calculated activity (Figure 53) of dilution.
2., for proving the OgLuc variant reactivity to plastics, 9B8 with L27V of purification is contacted with polystyrene plate, And measure its activity.
When 60,40,20 and 0 minutes, 0.1% will be contained without phenol redDMEM in 50 μ L purification 9B8 (45.3pM) and L27V (85.9pM) is placed in the hole of 96 hole polystyrene microtiter plate.Add containing 20 μMs of PBI-in sample The 50 μ L of 3939 measure reagent (as mentioned above) and at room temperature hatch 5 minutes.Measure luminescence as described earlier, and determine Percent activity (Figure 54；The luminous ratio with the time 0).
Embodiment 35 post translational modification
For determining whether OgLuc variant experiences any post translational modification, in suckling when expressing in mammalian cell Zooblast and Bacillus coli cells are expressed 9B8 and L27V variant, and is analyzed by mass spectrum (MS).
Using 9B8 and L27V variant as N-end in HEK293 cell and escherichia coli KRX (Promega Corp.) cell EndFusant (is pFN18K for Bacillus coli cells；It is pFN21K for HEK293 cell) express And utilizeProtein Purification System (Promega Corp.) is according to the explanation of manufacturer It is purified.Utilize the C4 post (Waters being connected with LTQ Orbitrap Velos mass spectrograph (Thermo Scientific) Xbridge BEH300,3.5 μm) enzyme of about 5 picomole purification is analyzed via LC/MS.Utilize for the LTQ detected from 600- 2000m/z obtains data and utilizes MagTran v1.03 software (Zhang etc., J.Am.Soc.Mass Spectrom., 9:225- 233 (1998)) process.With the 19 of the OgLuc variant (that is, there is not any post translational modification) of unmodified, the meter of 665Da The quality calculated compares, and the enzyme of two kinds of purification is respectively provided with 19 experimentally determined, the quality of 666Da.
Embodiment 36 OgLuc variant is as the assessment reporting son after transcribing
A.IV
Check that OgLuc variant is as the purposes reporting son after transcribing.For generating report after the transcribing of cAMP, utilize ability In territory, hRL and IV is subcloned into the pGL4 carrier of the modification containing barnase (barnase) sequence by known method In (Promega Corp.), described barnase sequence is substituted by DNA fragmentation interested.Before the pGL4 modified Lead sequence containing bottom line promoter and cAMP response element (CRE；SEQ ID NO:96), thus utilize cAMP agonist such as After forskolin (FSK) stimulates, the cell of accumulation cAMP makes reporter activation and generates luminescence.In this experiment, hRL or IV is used The 2ng DNA of the sub-construct of transcribed reporter transfected HEK 293 as described in embodiment 25.24 hours after transfection, use 100 μMs of FSK process cell.The cell that unused FSK processes is with comparing.After 6 hours, will report that sub-reagent adds process to Cell and compared with control cells.For hRL, report that sub-reagent is Renilla-GloTMReagent (Promega Corp.).For IV, report Accuse sub-reagent CDTApH5.5 Han 1mM, 150mM KCl, 10mM DTT, 0.5%NP-9 (v/v), 20 μMs of chambeies Intestinal element-h and 150mM thiourea.After 10 minutes,Luminescence is read on Flash (Thermo Scientific).
Figure 55 show containing with FSK process ("+FSK ") or unused FSK process ("-FSK ") hRL (" sea pansy genus ") or The standardized luminescence of the HEK293 cell of IV transcribed reporter.By being used for from the luminescence of the cell (+FSK) processed divided by coming Response is determined, i.e. luminous multiple induction or multiple increase (" multiple ") from the luminescence (-FSK) of compared with control cells.In Figure 55 Shown in, about hRL, response is ＜ 50, and it is ＞ 300 about IV, shows the IV purposes as transcribed reporter.
And 9B8opt B.9B8
Also check for variant 9B8 and 9B8opt as the purposes of transcribed reporter and as previously discussed with respect to the sub-institute of IV transcribed reporter Describe containing following amendment compared with hRL and Luc2 transcribed reporter.Generated as discussed above containing variant 9B8's or 9B8opt Transcribed reporter of cAMP.Induce after 6 hours at FSK, from cell, remove culture medium and with 100 described in embodiment 25 The lysis buffer of μ L is replaced and is generated lysate.Mensuration as described in embodiment 25 processes or without FSK process with FSK The luminescence of lysate of cell of transfection.BRIGHT-GLO with 50 μ LTMLuciferase measures reagent and measures 10 μ L's Luc2 lysate.The hRL lysate of 10 μ L is measured with the lysis buffer of the 50 μ L containing 20 μMs of natural coelenterazine.Utilize containing 20 μMs The 50 μ L lysis buffers of PBI-3939 measure variant 9B8 and the 9B8opt lysate of 10 μ L.
Figure 56 show (" substrate ") that process with FSK process (" induction ") or unused FSK containing 9B8,9B8opt, The standardized luminescence of the HEK293 cell of hRL or Luc2 transcribed reporter.By coming divided by substrate luminescence with the luminescence of induction Determine response, i.e. luminous multiple induction or multiple increase (" multiple ") (Figure 56).Although multiple induction value for except Luc2 it Outer each report is similar, 9B8opt transcribed reporter induced the luminescence generated belongs to transcribed reporter than the sea pansy of induction High about 2.5 logarithms of son, and than Luc2 transcribed reporter high about 1.5 logarithms of son.Figure 56 shows that 9B8 and 9B8opt is as transcribed reporter The purposes of son.
And 9B8opt+K33N C.9B8opt
Variant 9B8opt and 9B8opt+K33N is compared in cracking transcribed reporter measures.Utilize as known in the art Variant 9B8opt+K33N is cloned into containing ring-type AMP response element (CRE) pGL4.29 carrier (Promega Corp.) by method In.Inspection 9B8opt+K33N transcribed reporter is the most described above compared with 9B8opt transcribed reporter in HEK293 cell. The plasmid DNA of the 30 and 100ng of the use sub-form of transcribed reporter containing variant carrys out transfected HEK 293.Use before measuring luminescence FSK inducing cell 5 hours.With containing 1mM CTDA, 150mM KCl, 2mM DTT, 100mM MES pH6.0,35mM thiourea, 0.25%NP-9 (v/v) and the lysis buffer cell lysis of 10mg/mL2-HP-β-CD.?GENIOSTMLuminescence is measured on Pro photometer.Lysate is measured with the lysis buffer containing 20 μMs of PBI-3939. Figure 57 shows expression 9B8opt or 9B8opt+ of (" substrate ") of (" induction ") processed with FSK or unused FSK process The standardized luminescence (transfection correction) of the HEK293 cell of the sub-construct of K33N transcribed reporter.As shown in Figure 57, when with During the DNA transfection of 30ng, the multiple induction of 9B8opt is 360, and is 109 when transfect with 100ng, and 9B8opt+K33N again Number induction is respectively 275 and 147.When using the DNA transfection of higher amount, K33N provides higher response.
D.L27V
1. L27V is cloned into containing CRE, NFkB or HSE (Binding characteristic) response as described in the C of this embodiment In the sub-carrier of report of element.Then as described earlier reporter construct is transfected into HEK293 cell or HeLa cell In.Utilize the FSK about CRE, the TNF α about NFkB or the 17-AAG inducing cell about HSE.Utilize containing 20 μMs of PBI- Luminescence (Figure 58 A-C) measured as described earlier by the mensuration reagent of 3939.At HEK293, HeLa, NIH3T3, U2OS and Jurkat cell system demonstrates whole reporter construct (data are not shown).
2. as described in the C of this embodiment by L27V02 and L27V02P (containing PEST sequence；SEQ ID NO: 323) it is cloned in the sub-carrier of report (based on pGL4.32).Other OgLuc variants containing PEST sequence include L27V01- PEST00 and L27V03-PEST02 (respectively SEQ ID NO:320 and 326).Then sub-structure will be reported as described earlier Build body to be transfected in HEK293 cell.Then utilize FSK inducing cell, and utilize the mensuration reagent containing 20 μMs of PBI-3939 such as Previously described measurement is luminous (Figure 59 A-B).Also construct other different reporter construct and in different cell line In carried out checking (Figure 59 C).Figure 59 A shows the full dose response in HEK293 cell for CRE cell.Figure 59 B sums up Figure 59 B.Figure 59 C summarizes the data in Figure 59 A-B, and shows the identical data type for NFkB response element. In HEK293, HeLa, HepG2, Jurkat, ME180, HCT116 and U2OS cell line, checked CRE and NFkB report sub-structure Build both bodies.
3. utilizeHD (Promega Corp.) is according to the explanation of manufacturer pNFkB-L27V secreting type Construct (SEQ ID NO:463&464；Wherein IL-6 secretion sequence (SEQ ID NO:461 and 462) instead of natural OgLuc Secretion sequence SEQ ID NO:54), Metridia longa (Clontech), pNFkB-L27V (natural secretion sequence；SEQ ID NO:465 and 466) or LUC Photinus pyralis LUC Photinus pyralis FL (Luc2；Based on pGL4.32) plasmid DNA transfection HEK293 cell is (in T25 flask 0.9x106Individual cell).At 37 DEG C, 5%CO2Lower incubated cell 8 hours, then pancreas in 0.5mL TrypLE (Invitrogen) Enzymic digestion.Then lysate is resuspended in the 8mL DMEM containing 10%FBS, 1X NEAA and 1X Sodium Pyruvate.Then by 100 μ The sample of the gravity treatment of L adds in the hole of 96 hole flat boards and at 37 DEG C, 5%CO2Under hatch 16 hours.
After hatching, from cell, remove culture medium and with containing TNF α or 100 μ L new without TNF α (serial dilution) Fresh culture medium substitutes.For measuring secretion, little constantly 3 hours and 6, the culture medium (three repetitions) of 5 μ L is removed from cell, PBS is utilized to increase to 50 μ L the mensuration reagent (as described earlier containing 100 μMs of PBI-3939) with 50 μ L.Such as previous institute Describe measures luminous (Figure 60) when 0 minute and 10 minutes.
For measuring elongated long ascites flea uciferase activity, use Ready-To-according to the scheme of manufacturer GlowTMSecreted Luciferase System(Clontech).In short, by 5 μ L Ready-to-GlowTMReagent adds It is added in the PBS of 5 μ L sample and 45 μ L.Luminescence (Figure 60) is measured immediately after adding reagent.
The variant that E.L27V optimizes.
Scheme according to manufacturer utilizesHD preparation is for the plasmid DNA (pGL4.32-of transfection L27V00, pGL4.32-L27V01, pGL4.32-L27V02, pGL4.32-L27V03 and pGL4.13).PGL4.32 carrier (Promega Corp.) κ B response element Han NF-.Luc2P sequence in the sequence replacing that L27V is codon optimized carrier. The pGL4.13 carrier (Promega Corp.) the Luc2 gene containing SV40 promoters driven.
Then by the HeLa cell suspending liquid (2x10 of the DNA transfection mixture of 300 μ L Yu 6mL5Individual cell/mL) mix mutually Close, homogenize, and 100 μ L are inoculated in the hole of 96 orifice plates.Then at 37 DEG C, 5%CO2Lower night incubation cell.After hatching, 10X rhTNF α in the 10 μ L DPBS containing BSA is added in hole and at 37 DEG C, 5%CO2Under hatch 4.5 hours.6 holes are only As carrier.Then make cell at room temperature balance 20 minutes, and then add 100 μ L mensuration reagent (as described previously Containing 100 μMs of PBI-3939).The ONE-of 100 μ L is added in expressing the cell of Luc2 or only accepting the cell of vehicle treated GLOTMLuciferase Assay Reagent.Luminescence is measured as described earlier after adding mensuration reagent 12 minutes.Figure 61 A-B shows Absolute luminescence, Figure 61 C-D shows that standardized luminescence and Figure 61 E-F show multiple response.
OgLuc variant in embodiment 37 transcribed reporter mensuration
For prove the present invention OgLuc variant be used as transcribed reporter ability, by OgLuc variant 9B8opt forward, Reversely and batch transfection is used as transcribed reporter.These are selected to transfect the scheme according to manufacturer, because which represent gene Method conventional in the transient expression of transcribed reporter.
Forward transfects
PGL4.29 (Promega Corp.) skeleton is prepared for containing cAMP response element (CRE) and 9B8opt or also wraps Include transcribed reporter of the 9B8opt (9B8opt-P) of PEST protein degradation sequence, i.e. the luc2P gene quilt of pGL4.29 carrier 9B8opt (SEQ ID NO:24) or 9B8opt-P (SEQ ID NO:65) is substituted.PGL4.29 is with comparing/datum mark.
HEK293 cell is seeded in 6 96 hole tissue culture plate with 15,000 cells/well.100 μ L's DMEM+10%FBS+1X non essential amino acid (NEAA) is cultivated cell and at 37 DEG C overnight incubation.Utilize 10ng or the 100ng plasmid DNA of pGL4.299B8opt, pGL4.299B8opt-P or pGL4.29/hole transient transfection cell.Will Plasmid DNA is with 850 μ L's(Invitrogen) and 32.4 μ L'sHD transfection reagent (Promega Corp.) mixes mutually and at room temperature hatches 10 minutes.By the transfection of 8 μ L/report that sub-DNA mixture adds to suitable When hole (2 construct/flat boards).Incubated cell 4 hours at 37 DEG C.WithThe FBS+ of+0.5% dialysis 1X NEAA+1X Sodium Pyruvate+1X Pen .-Strep replace culture medium and at 37 DEG C night incubation.
After hatching, 10nM or 10 μMs of FSK (from 10X stock solution) will be containedAdd in hole and 3 hours are hatched at 37 DEG C.To contain 100mM MES pH6.1,1mM CDTA, 150mM KCl, 35mM thiourea, 2mM DTT, 0.25%NP-9 (v/v), 0.025%The lytic reagent of DF204 and 20 μMs of PBI-3939 adds It is added in the cell containing pGL4.299B8opt or pGL4.299B8opt-P and at room temperature hatches (100 μ L cracking examinations in 10 minutes Agent is added in 100 μ L cells).By ONE-GLOTMMeasure reagent (Promega Corp.) to add in the cell containing pGL4.29 And use (100 μ L reagent add 100 μ L cells to) according to the scheme of manufacturer.?Measure on photometer Luminous.Table 26 shows that the HEK293 expressing the transcribed reporter containing CRE processed with 10nM (" baseline ") or 10mM FSK is thin The luminescence of born of the same parents, and to the response of FSK (that is, 10mM FSK the luminescence of the Hemapoiesis processed divided by 10nM FSK process thin The luminescence that born of the same parents generate).
In table 28, the result of display shows that 9B8opt and 9B8opt-P is brighter than luc2P, and when the DNA using 100ng enters During row transfection, all Luciferase reporter are to FSK response.But, when the DNA only using 10ng transfects, luc2P's The luminous detection level less than luminometer.
In table 28:HEK293 cell, the transcribed reporter containing CRE is sub (3 hours point)
Reverse transfection
The scheme according to manufacturer as known in the art is utilized to prepare in pGL4.29 (Promega Corp.) skeleton Transcribed reporter containing antioxidant response element (ARE) and 9B8opt or 9B8opt-P, i.e. the luc2P base of pGL4.29 carrier Because being substituted by 9B9opt or 9B8opt-P, and CRE is substituted by 2X ARE (SEQ ID NO:66).
With trypsinization HEK293 cell (T75 flask, 3mL pancreatin) and with 1x105Individual cell/mL (about 8.9x106Individual always Cell) it is resuspended in the culture medium containing DMEM+10%FBS+1X NEAA.By by 1.2mL12 μ L transcribe Report sub-DNA (100ng) and 36 μ LHD transfection reagent is mixed together to prepare every kind for transfection and transcribes Report, and at room temperature hatch 35 minutes.After hatching, by the transfection of 624 μ L/report that sub-DNA mixture adds 12mL's to Mix in cell suspending liquid and by overturning.After mixing, add the cell/DNA mixture of 100 μ L to 96-hole flat board In hole (2 construct/flat boards).Incubated cell 22 hours at 37 DEG C.Tert-butyl hydroquinone (Nrf2 stabilizer will be contained； tBHQ；1 μM (" baseline ") or 20 μMs) or sulforaphen (the organic sulfide antioxidant of known activation Nrf2；1 μM (" baseline ") or 20 μMs)Add every hole to and hatch at 37 DEG C 24 hours.As described above for the utilization described in forward transfection The lytic reagent cell lysis of 100 μ L.?Luminescence is measured on photometer.
Table 29 shows the expression utilizing 1 μM (" baseline ") or 20 μMs of sulforaphen process transcribed reporter containing ARE The luminescence of HEK293 cell and to the response of sulforaphen (that is, by removing with the luminescence of the Hemapoiesis processed by 1 μM of sulforaphen The luminescence of the Hemapoiesis to be processed by 20 μMs of sulforaphen).Table 30 shows and utilizes 1 μM (" baseline ") or 20 μMs of tBHQ process The HEK293 cell expressing transcribed reporter containing ARE luminescence and to the response of tBHQ (that is, by with by 1 μM of tBHQ The luminescence of the Hemapoiesis of reason is divided by the luminescence of the Hemapoiesis processed by 20 μMs of tBHQ).Table 29 and 30 show 9B8opt and 9B8opt-P can report the existence of the stimulus object of two kinds of different known ARE.
In table 29:HEK293 cell, the transcribed reporter containing ARE is sub (24 hours point)
In table 30:HEK293 cell, the transcribed reporter containing ARE is sub (24 hours point)
Batch transfection
By forward transfect described in transcribed reporter containing CRE and 9B8opt or 9B8opt-P be used for HEK293 and In the batch transfection of NIH3T3 cell.The element Han Heat shock response is prepared in pGL4.29 (Promega Corp.) skeleton (HRE；SEQ ID NO:67) and transcribed reporter of 9B8opt or 9B8opt-P, i.e. substitute with 9B9opt or 9B8opt-P The luc2P gene of pGL4.29, and substitute CRE with HRE.Transcribed reporter containing HRE and 9B8opt-P is used for HeLa cell In batch transfection.
At day before transfection, HEK293, NIH3T3 or HeLa cell is inoculated into the single hole of 6 hole tissue culture plate, Density is for be contained in 3mL complete medium (DMEM+10%FBS+1X NEAA+1X Sodium Pyruvate) for HEK293 cell 4.5x105Individual cells/well, for NIH3T3 cell for being contained in 3mL complete medium (DMEM+10% hyclone (FCS)+1X NEAA+1X Sodium Pyruvate) in 3x105Individual cells/well, or for HeLa cell for being contained in 3mL complete medium (DMEM+ 9.9x10 in 10%FBS+1XNEAA)5Individual cells/well.Cell is grown overnight at 37 DEG C.
By 155 μ LIn the reporter plasmid DNA and 9.9 μ L of 3,300ngHD turns Transfection reagent mixes mutually, simple vortex, and at room temperature hatches 10 minutes.Use CRE transcribed reporter transfect HEK293 and NIH3T3 cell.HRE transcribed reporter is used to carry out transfection HeLa cell.Sub-for report mixture is added in cell and by light Micro-shake mixes, and then hatches 6 hours (HEK293 and NIH3T3) or 3 hours (HeLa) at 37 DEG C.Then trypsinization Cell to be resuspended in culture medium (be DMEM+10%FBS+1X NEAA+1X Sodium Pyruvate for HEK293 cell, for NIH3T3 Cell is DMEM+10%FCS+1X NEAA+1X Sodium Pyruvate or is DMEM+10%FBS+1X NEAA for HeLa cell), so After be inoculated into the single hole of 96 hole flat boards and (be 20,000 cell/100 μ L for HEK293, be 10,000 for NIH3T3 Individual cell/100 μ L, or be 13,000 cell/μ L for HeLa) and at 37 DEG C overnight incubation.
FSK (CRE stimulant) or 17-AAG (HRE stimulant will be contained；17-allylamino-demethoxygeldanamycin) 'sAdding cell to (is 10nM or 10 μMs of final concentrations for FSK；For 17-AAG be 1nM or 1 μM of end is dense Degree 17-AAG) in and hatch 4 hours (FSK) or 6 hours (17-AAG) at 37 DEG C.From couveuse, remove flat board and make it balance To room temperature, continue 25 minutes.As described above for described in forward transfection with 100 μ L lytic reagent cell lysis.?Luminescence is measured on photometer.
Table 31 shows expresses, with what 10nM (" baseline ") or 10mM FSK processed, the HEK293 that the transcribed reporter containing CRE is sub The luminescence of cell, and the response to FSK.Table 32 shows and expresses containing CRE's with what 10nM (" baseline ") or 10mM FSK processed The luminescence of the NIH3T3 cell of transcribed reporter, and the response to FSK.Table 33 shows with 10nM (" baseline ") or 10mM17- What AAG processed expresses the luminescence of the HeLa cell of transcribed reporter containing HRE, and the response to 17-AAG.
Table 29-31 shows 1) in the environment of two kinds of different cell lines HEK293 and NIH3T3,9B8opt OgLuc Two kinds of forms of variant can report existence and the FSK stimulation to CRE of FSK, and 2) in the environment of HeLa cell, 9B8optP can report existence and the 17-AAG stimulation to HRE of 17-AAG.
In table 31:HEK293 cell, the transcribed reporter containing CRE is sub (4 hours point)
In table 32:NIH3T3 cell, the transcribed reporter containing CRE is sub (4 hours point)
In table 33:HeLa cell, the transcribed reporter containing HRE is sub (6 hours point)
Report with secretion of the cracking in the cell that embodiment 38 is beyond expression of words
Explanation according to manufacturer utilizesHD utilize containing L27V02, luc2P (Promega Corp.), Luc2 (Promega Corp.) or the L27V02-IL6 (L27V02 containing the natural secretion sequence substituted by IL-6 secretion sequence； (“IL601-L27V02A”；SEQ ID NO:324) plasmid DNA (pGL4.32 skeleton, Promega Corp.) to cell suspension 1x10 in liquid5The HepG2 cell of individual cell/mL inversely transfects (1: 20DNA-transfection mixture is than cell).Then will 100 μ L cell suspending liquids are inoculated in the hole of 96 orifice plates and at 37 DEG C, 5%CO2Under hatch 22 hours.Other have and are divided by IL-6 The OgLuc construct of the natural secretion sequence secreting sequence replacing includes IL601-L27V01 and IL602-L27V03 (respectively SEQ ID NO:321 and 327).
Secretion is analyzed, from cell, removes culture medium, and washed cell in 100 μ L DPBS.Add 100 μ L complete Full culture medium (DMEM+10%FBS+1X NEAA) and the rhTNF α (" TNF α ") of various dose (1pg/mL-100ng/mL), hold Continuous 4.5 hours.Remove the culture medium of 10 μ L, be added to the complete medium of 90 μ L, and add 100 μ L mensuration reagent (as Previously described；100μM PBI-3939).Measure luminescence (Figure 62 A) as described earlier.
For cracking analysis, after inoculation, by cell at 37 DEG C, 5%CO2Under hatch 4.5 hours.Then cell balance is made To room temperature, continue 20 minutes.Add to cell and measure reagent (as described earlier；100 μMs of PBI-3939), and as previously Described measurement is luminous (Figure 62 B).
Embodiment 39 other cracking report subcharacter
In environment based on cell, the OgLuc variant of the present invention, cracking transcribed reporter should provide sending out of some strength Optical signal thus signal occur to occur faster with other luciferases than it.This bright burn allows also to check weak opening Mover.
Embodiment 40 mammalian cell transfection
The OgLuc variant of the present invention in being difficult to the cell line transfected with son of giving a report, such as, Jurkat, HepG2, former For cell, nondividing primary cell or stem cell (seeing, Figure 59 C).Due to its high signal intensity, when transfection efficiency is low, OgLuc variant can make luminescence to detect.OgLuc variant also act as especially to the relevant condition of transfection (that is, DNA concentration, Transfection reagent adds) report in sensitive cell.Due to the brightness of OgLuc variant, utilize relatively low DNA concentration, less Transfection reagent and after shorter may starting the transfection before measuring the time and the luminescence of enough levels can be obtained.This will make sensitivity The toxicity burden that type cell may bear is less.In all events if desired for output, the bright burn of OgLuc variant is also assented Permitted at very long time point detection signal.As another example, OgLuc variant possibly serves for for single copy natural promoter Report son, such as, HSB thymidylate kinase (TK) promoter, hox gene or LIN28.
The cell line that embodiment 41 is stable
The bright signal of luciferase and the small size of OgLuc gene can beneficially express the present invention Cytoplasm form or Firm, the qualification of stable cell lines of the OgLuc variant of secreted form.This relatively small gene order should reduce due to external source The probability of the genic instability that the integration of DNA causes.
For utilizing the OgLuc variant of the present invention to generate stable cell line, use and include OgLuc variant and selective key The plasmid DNA of the nucleotide sequence of thing gene (such as, neomycin, hygromycin or puromycin) transfects cell interested System, such as, HEK293 cell.By the cell of early stage number of passages, such as, below 10 generations, it is inoculated into T25 (1x106) or T75 (3x106) in tissue culture flasks and allow its growth overnight to converge to about 75%.Then above plasmid DNA and suitable is utilized Transfection reagent, such as,OrHD carrys out transfectional cell.Transfect latter 48 hours, with containing previously Determine that the Selective agar medium selecting medicine such as G418, hygromycin or puromycin of the concentration of the cell killing untransfected is replaced Cell culture medium.The cell containing plasmid DNA is selected after 2-4 week.During this period, with different concentration, cell is inoculated T25 Or in the Selective agar medium of T75 tissue culture flasks.The training on the cell inoculated within every 3-4 days, is replaced with fresh selection medium Support base, continue 2-3 week.The formation of the living cells clone of monitoring flask.Finally, flask is by containing many large-scale clones and few dead Cell.
In the mixture of the stable clone from flask, separate monoclonal and expand single 24 hole tissue culturing plates In.In short, utilize pancreatin/EDTA method harvesting, i.e. by removing culture medium, with without Ca2+And Mg2+PBS drift Wash and by separating and harvesting with pancreatin/EDTA process.Utilize hemocytometer to count cell and by 1x105It has been diluted in In full culture medium.Then cell is diluted in complete medium 100 cell/mL, 33 cells/mL, 10 cell/mL and 3.3 cell/mL.Each diluent of 100 μ L is inoculated into 96 hole tissue culture plate institute porose in (each diluent 1 Flat board) and allow growth 4-5 days, add the Selective agar medium of 50 μ L to cell afterwards.After inoculating about 1 week, range estimation screening is thin The clonal growth of born of the same parents also adds the Selective agar medium of another 50 μ L.Continue monitoring cell until monoclonal covers the 40-of hole area 60%.When clone will expand and screen, utilize pancreatin/EDTA method results clone.Following being transferred to by each clone is selected Select in culture medium: 1) it is diluted to for 6 holes of 96 hole assay plate of functional examination (such as, luminous detection) at 1: 10；2)1∶ 10 are diluted to measure (such as, for cell survival Luminescent Cell Viability Assay (Promega Corp.)) clear bottom 96 hole assay plate 3 holes in；3) 1: 10 be diluted to for In 24 hole tissue culture plate of amplification.Then the cell in the flat board being used for functional examination and cell survival mensuration is cultivated 2-3 days and carry out functional examination and cell survival and measure.Then functional examination and cell survival is utilized to measure inspection further Test the positive colony in 24 hole flat boards and check stability and response at least 20 generation of expression, normal growth rate form, and Freezing when may pass on the earliest is for using in the future.
Embodiment 42 OgLuc secretion signal is analyzed
A.IV opt
In post synthesis wild type OgLuc is processed as the maturation protein that secretory signal sequence cracking is fallen.For determining this secretion Whether signal sequence will assist in the secretion of OgLuc variant, is cloned into IV opt variant and the hRL of embodiment 25 containing N-end In the pF4Ag of OgLuc secretion signal (SEQ ID NO:54).Utilize the plasmid DNA of 100ng, i.e. with or without secretion signal The transfection as described in embodiment 25 of the plasmid DNA of hRL or the IV opt 100 μ L Da Er containing 10% hyclone (FBS) Bai Ke (family name) improves the HEK293 cell (15,000) in Iger (family name) culture medium (" DMEM ") and grows overnight at 37 DEG C. The culture medium of 50 μ L it is moved out in new flat board and preserves for mensuration after a while, generating " culture medium " sample.Remove residue Culture medium, and generate " lysate " sample with the lysis buffer cell lysis of 100 μ L described in embodiment 25.Measure 10 μ L media samples and the luminescence (Figure 63) of 10 μ L lysate samples.Utilize the 50 μ L cracking bufferings containing 20 μMs of natural coelenterazine Liquid is measured containing OgLuc secretory signal sequence (" Renilla sig ") or without OgLuc secretory signal sequence (" Renilla ") The sample of hRL.The 50 μ L lysis buffers containing 20 μMs of PBI-3939 are utilized to measure (" the IV opt Han OgLuc secretory signal sequence Sig ") or the sample of IV without OgLuc secretory signal sequence (" IV opt ").
In Figure 63, the light that solid bars representative detects in the case of there is not any lytic reagent from culture medium Amount.Hollow strips represents at the total light (secretion+non-secretory) adding detection after lytic reagent.Figure 63 show IV opt from HEK293 cell is secreted in growth medium and secretory signal sequence is functional in mammalian cell.“IV Opt sig " represent the unique situation significantly measured luciferase being detected in the medium.Result also shows that this is specifically believed Number peptide will not assist the secretion of hRL.
B.9B8, V2 and L27V
For determining whether the secretory signal sequence of OgLuc assists it to secrete, OgLuc variant 9B8, V2 and L27V are cloned into In pF4Ag containing N-end OgLuc secretory signal sequence.Also by variant clone to without in the carrier of secretory signal sequence.So After CHO or HeLa cell is inoculated into 100,000 cells/well the 1mL containing 10%FBS and 1X Sodium Pyruvate of 12 hole flat boards F12 culture medium (Chinese hamster ovary celI) or containing 10%FBS and 1X Sodium Pyruvate DMEM (HeLa cell) in and at 37 DEG C, 5%CO2 Lower overnight incubation.
After night incubation, utilizeTransfection reagent (Mirus Bio) andCultivate Base (Invitrogen) utilizes the 1 μ g DNA transfectional cell containing 9B8, V2 or L27V with and without secretory signal sequence.Again Secondary by cell at 37 DEG C, 5%CO2Lower overnight incubation.
For the second time after night incubation, removal culture medium also preserves for analysis.The mensuration adding 1mL in cell is delayed Rush liquid (1mM CDTA, 150mM KCl, 2mM DTT, 100mM MES pH6.0,35mM thiourea and 0.5% NP-9 (v/v)) with cellulation lysate.Add in the cell lysate of 10 μ L or the culture medium of preservation of each sample Add the mensuration buffer containing 40 μMs of PBI-3939 of 50 μ L, and luminescence measured as described above.Figure 64 A-D show 9B8, V2 and L27V variant can be used for can excretory system.
For determining the stability of the variant of secretion, the culture medium of the preservation of the 150 μ L aliquot from each sample is placed in At 37 DEG C or 50 DEG C.Then at different time point (0,1,2,3,5,6 and 7 minute) removal equal portions, freezing on dry ice, and Preserve until measuring at-20 DEG C.For measuring stability, culture medium equal portions are melted to room temperature, and every by 10 μ L as mentioned above Individual equal portions and the mensuration buffering liquid-phase mixing containing PBI-3939 (pH6.0).Luminescence measured as above, and determine half-life (t50) (table 34)。
Table 34
C.9B8 with V2 about the comparison of the luciferase of the secretion of elongated long ascites flea
OgLuc variant 9B8 and V2 compares about the secretion of the luciferase of the secretion from elongated long ascites flea.Will In the 3mL F12 culture medium containing 10%FBS that Chinese hamster ovary celI is inoculated in the hole of 6 hole flat boards with 300,000 cells/well and 37 DEG C, 5%CO2Lower night incubation.Then utilize according to the explanation of manufacturerUtilize 10 or 100ng Each variant or long abdomen Daphnia luciferase (Clontech) plasmid DNA transfection cell, and at 37 DEG C, 5%CO2Under hatch 20 Hour.After transfection, from cell, remove culture medium and measure.For OgLuc variant, utilize mensuration reagent (the previous institute of 50 μ L Describe；40 μMs of PBI-3939) measure the culture medium of 50 μ L.For long abdomen Daphnia luciferase, according to the scheme of manufacturer Utilize Ready-to-GloTMSecreted Luciferase Reporter System (Clontech) measures culture medium.Letter speech It, add to the 1X substrate/reaction buffer of 5 μ L in the media samples of 50 μ L.Then as described earlier measurement is sent out Light (Figure 65 A-B).
OgLuc variant in embodiment 43 living cells and the assessment of novel coelenterazine
A. the OgLuc variant in living cells and the purposes of PBI-3939 are checked.By HEK293 cell with 15,000 cells/ Hole is seeded in 96 hole flat boards and at 37 DEG C and grows overnight.Second day, utilize the pF4Ag containing hRL or 9B8opt of 100ng with Three recyclingsTransient transfection cell also grows overnight at 37 DEG C.Within second day, remove grown cultures Base with containing 60 μMs of VIVIRENTMLive Cell Substrate (Promega Corp.), 60 μMs of ENDURENTMLive Cell Substrate (Promega Corp.) or 60 μMs of PBI-3939 of the cell for hRL and 9B8opt transfection Culture medium substitutes.The cell of untransfected is used as ground control.Hatched at 37 DEG C during one day flat board andGENIOSTMPeriodically measure on Pro photometer, i.e. carried out during 24 hours 11 times.Figure 66 A-B Show that the luminescence of the cell of transfection is divided by the luminescence of the cell of untransfected for every kind of substrate, i.e. signal compares background rate. Data show by with VIVIRENTM, ENDURENTMOr in PBI-3939 incubated cell living cells background (that is, uncracked) The luminescence that 9B8opt generates.Data also show that PBI-3939 can permeate the cell in cultivation, react with OgLuc variant and generate Luminescence, so that the purposes during it measures with living cells is compatible.
B. for utilize OgLuc variant prove live cell assays, by L27V withMelt and be incorporated in living cells Express and monitoring.U2OS cell is inoculated in imaging chamber hole with 40,000 cell/mL and at 37 DEG C, 5%CO2Under overnight incubate Educate.Then plasmid pFC14K, pFN21K or the pF4Ag (being Promega Corp.) containing L27V or natural containing having is utilized Or the pF4Ag of the L27V of IL-6 secretion sequence utilizes according to the scheme of manufacturerHD transfectional cell.Then 37 DEG C, 5%CO2Lower incubated cell 24 hours.
After hatching, by cell withTMR part (Promega Corp.) contacts, and imaging is also fixed.So Basis afterwardsICC scheme in technology: focal imaging technical manual (Promega Corp.；TM260) carry out Immunocytochemistry (ICC).One used resists for multi-clone rabbit, anti-OgLuc9B8 antibody (1: 1000).Use two resist for The two anti-Alexa488 (green) (Figure 67 A) puted together.Figure 67 A shows that green fluorescence channel and Figure 67 B show that differential is interfered Difference (DIC).Utilization is equipped with the Olympus of 37 DEG C of+CO2 environmental chambers (Solent Scientific Ltd., UK) Fluoview FV500 Laser Scanning Confocal Microscope (Olympus, USA) obtains image.
Figure 67 B-D shows the ICC image with natural or IL-6 secretion sequence.Two kinds of signal sequences significantly increase The amount of the enzyme in core.The character emphasized of the labelling in Cytoplasm represents it is contemplated that the vesicle shape that occurs in secretion process Become.The existence of tables of data clear signal peptide reduces the amount of the luciferase in core.
C. as it appears from the above, the OgLuc variant of the present invention and novel substrate are biocompatible.Contemplate wherein by OgLuc Variant clone is to report expressed as reporting sub-albumen in the expression vector containing promoter interested and in cell System.Then utilize the PBI-3939 of the cell in infiltration cultivation to process cell, make cell react with OgLuc variant, and Generate luminescence.
In addition to as Premeabilisation of cells thing, PBI-3939 shows suitable with natural coelenterazine in terms of cell survival Biocompatibility.The compound containing chemical modification of the known stability improving natural coelenterazine in the medium can be synthesized A kind of form of 3939, and for consolidate, report based on living cells OgLuc variant measures.Living cells report another Example includes using the OgLuc variant that can secrete as report.Can be by (or other the known secretions of natural secreting signal peptide Signal peptide) with the N-terminal fusion of OgLuc variant thus when fusant is expressed in mammal, its part will pass through cell Film is secreted in culture medium.Luminescence is generated after adding substrate.
Embodiment 44 protein fusion report
The OgLuc variant of the present invention can be used as the fusion tag of target protein interested, as monitoring the thin of this target protein The means of intracellular level.Can monitor the specific albumen participating in stress response approach in cell, such as, DNA damage, oxidation should Swash, inflammation, the means that may act on played in these approach as the stimulus object detecting dissimilar terms.Variant also acts as prison Survey the instrument of the cell transport of target protein.Variant also can merge thus with potential with viral genome (such as, HIV, HCV) Antiviral agent can monitor titre levels after processing in cell, i.e. infectivity.Variant also can with green fluorescent protein (GFP) or(in addition to target protein) merges thus FACS can be used for identifying high-expression clone and providing location information.
The assessment of the OgLuc variant in embodiment 45 3-assembly fusion protein (" sandwich ")
3-component fusion protein or " sandwich " fusant, can be used for being placed in mutually bioluminescent protein and fluorescin Near to optimize biosensor based on BRET.
A.C1+4AE, IV, 9B8 and 9F6
Utilize be known as poor fusion partner N-end Id (Benezra etc., Cell, 61 (1): 49-59 (1990)) and For standardized C-end HT7 by OgLuc variant C1+4AE (SEQ ID NO:55 and 56), IV (SEQ ID NO:57 and 58), 9B8 (SEQ ID NO:61 and 62) and 9F6 (SEQ ID NO:63 and 64) and hRL (SEQ ID NO:32 and 33) is cloned into In pF4Ag fusion vector.By gene interested between Id and HT7 (that is, Id-luciferase-HT7) " sandwich ", as The preparation described in embodiment 26 E. coli lysate containing the variant construct in pF4Ag or pF4Ag sandwich background, And be then measured by 20 μMs of natural coelenterazine in the buffer described in embodiment 25.
Figure 68 shows the luminescence of each variant in pF4Ag or pF4Ag background (" sandwich ").Figure 69 show due to The luminous multiple of the existence of Id and HT7 increases, and by with the luminescence of the variant in pF4Ag divided by the change in pF4Ag-sandwich The luminescence of body determines.The sample with maximum shows the strongest sensitivity for poor fusion partner Id.Variant 9B8 It is the brightest in sandwich environment.
And 9B8OPT+K33N B.9B8OPT
Analysis variant 9B8opt and 9B8opt+K33N in sandwich background as described earlier.As described previously Generation 9B8opt (SEQ ID NO:40 and 41) and the sandwich construct of 9B8opt+K33N (SEQ ID NO:59 and 60). Utilize and be used for generating the identical mensuration buffer used by Figure 40 and photometric determination and measuring E. coli lysate.Figure 70 show that the multiple in the presence of sandwich background increases, and show that 9B8opt+K33N is not so good as 9B8opt to poor fusion partner Id is more sensitive.
And 24C2 C.23D2
Variant 23D4 (NF) and 24C2 (NF) is subcloned in Id-OgLuc-HT7 sandwich background and escherichia coli In be measured.By sandwich variant 23D4 (F) (SEQ ID NO:76 and 77) and 24C2 (F) (SEQ ID NO:78 and 79) with 9B8opt+K33N (SEQ ID NO:59 and 60) in sandwich background compares.Table 35 shows at sandwich background environment Middle variant has the luminescence the most identical with 9B8opt+K33N.
Table 35: the luminescence generated by OgLuc variant increasing compared with the 9B8opt+K33N+170G in sandwich background Add
And 15H1 D.1F7
Screening is the literary composition of the PCR in Id-OgLuc-HT7 sandwich background compared with the 9B8opt+K33N in sandwich background The other variant with the luminescence increased in storehouse.Then in HEK293 cell and NIH3T3 cell, measure the variant of selection. Table 36 show the luminous multiple of in Bacillus coli cells, HEK293 cell and NIH3T3 cell sandwich variant increase and The amino acid replacement found in variant.1F7 (F) (SEQ ID NO:84 and 85) and 15H1 (F) (SEQ ID NO:86 and 87) There are in escherichia coli at least 1.3 times of luminescences increased.In HEK293 cell and NIH3T3 cell, 1F7 (F) ratio is in Sanming City Control the 9B8opt+K33N in background brighter.
Table 36: the luminescence generated by OgLuc variant increase compared with the 9B8opt+K33N in sandwich background
By in sandwich variant clone to non-fused background carrier based on pF4Ag with generate 1F7 (NF) (SEQ ID NO: 80 and 81) and 15H1 (NF) (SEQ ID NO:82 and 83) being analyzed as described earlier, and with 9B8opt+K33N phase Relatively.Table 37 shows that the luminous multiple of variant increases in Bacillus coli cells, HEK293 cell and NIH3T3 cell.? In escherichia coli and HEK293 cell, 1F7 (NF) and 15H1 (F) has the luminescence that at least 1.3 multiples increase.
Table 37: the luminescence generated by OgLuc variant increasing compared with the 9B8opt+K33N+170G in sandwich background Add
E.V2,9B8opt+K33N+L27V+K43R+Y68D, 9B8opt+K33N+L27V+T39T+K43R+S66N and L27V
As described earlier by variant 9B8opt+K33N+T39T+K43R+Y68D (" V2 "；SEQ ID NO:92 and 93), 9B8opt+K33N+L27V+K43R+Y68D (SEQ ID NO:339 and 340), 9B8opt+K33N+L27V+T39T+K43R + S66N (SEQ ID NO:341 and 342) and 9B8opt+K33N+L27V+T39T+K43R+Y68D (" L27V "；SEQ ID NO: 88 and 89) it is subcloned in Id-OgLuc-HT7 sandwich background and as described earlier in HEK293 and NIH3T3 cell It is measured.The luminescence generated by sandwich variant is generated with by 9B9opt+K33N sandwich (SEQ ID NO:59 and 60) Luminescence compare (table 38).L27V sandwich (SEQ ID NO:90 and 91) and V2 sandwich (SEQ ID NO:94 and 95) exist HEK293 cell and NIH3T3 cell have the luminescence that at least 1.3X multiple increases.
Table 38: the luminescence generated by the OgLuc variant in sandwich background and the 9B8opt+K33N phase in sandwich background Increase relatively
Mensuration variant V2,9B8opt+K33N+ in HEK293 cell and NIH3T3 cell as described in embodiment 37 The sandwich of L27V+K43R+Y68D, 9B8opt+K33N+L27V+T39T+K43R+S66N and L27V and non-sandwich formats.Will The luminescence (table 39) compared with the luminescence generated by sandwich variant generated by the variant of non-sandwich.Number shown in table 39 Reduce in mammalian cell than in Bacillus coli cells more according to the luminous multiple showing 9B8opt+K33N sandwich Low, as shown in Figure 70.
Table 39: the luminous multiple of OgLuc variant reduces in the presence of sandwich background
Embodiment 46 MULTIPLE COMPOSITE
A. as previously preparation described in embodiment 27 express variant 9B8opt colibacillary lysate and Without phenol red+0.1%DMEM in dilute 1000 times.Utilize amendment The red Pleonomus of the purification that Luciferase Assay System (Promega Corp.) detection carrys out self-contained 6.3 μ g/mL is glimmering The luminescence of the E. coli lysate of the luminescence of the sample of light element enzyme and expression variant 9B8opt.According to the scheme of manufacturer, make With containing 20 μMs of coelenterazine-h'sSTOP&Reagent and containing 20 μMs of PBI3939 Reagent detects the red Pleonomus luciferase from single sample With OgLuc variant 9B8 luciferase.Carry out three repetitions.
At Turner MODULUSTMDetection luminescence on photometer.Table 40 shows (" is kowtowed by red Pleonomus luciferase Cephalont ") average luminescence that generates, and utilized coelenterazine-h (" coelenterazine h ") or PBI-3939 by 9B8opt (" OgLuc ") The luminescence that (" 3939 ") generate.Also show standard deviation (" +/-") and the coefficient of variation (" CV ").Carry out " without coelenterazine " comparison Be shown in do not exist in the case of coelenterazine byLuciferase Assay System'sThe amount of the red Pleonomus signal of Reagent institute cancellation." without coelenterazine " compares Create 349 times of cancellation.Table 40 show in simple sample detection from red Pleonomus and OgLuc variant 9B8 Large-sized luminous signal.This shows that every kind of signal can read in two pacings are fixed in succession, and can be by foot from the signal of the first enzyme Enough cancellation are without interfering significantly on the signal from the second enzyme.
Table 40: utilize the DUAL-LUCIFERASE of amendmentTMReport measures by red Pleonomus and 9B8opt luciferase The average luminescence generated
B. for proving that MULTIPLE COMPOSITE as above report measures and can be reversed, i.e. first detection OgLuc is luminous, Cancellation also detects the second luminescence, such as, and red Pleonomus or luciferase luciferase, screened multiple sea pansy and belonged to luciferase Inhibitor (seeing U.S.'s published application the 2008/0248511st) suppresses the ability of OgLuc equally.Different previous by two kinds The sea pansy identified belongs to inhibitor PBI-3077 and 1424 and adds the large intestine expressing variant 9B8 with different concentration (seeing table 41) to Bacillus lysate sample (as above diluting) and the MES pH6.0 Han 100mM, 1mM CDTA, 150mM KCl, 35mM thiourea, 2mM DTT, 0.25%NP-9 (v/v), 0.025%DF204 and the buffer of 20 μMs of PBI-3939 In.Measure luminescence as described earlier, except for the difference that utilizeMicroplate photometer (Promega Corp.；Also referred to as Turner MODULUSTM) measure luminescence.As shown in table 41, two kinds of compounds all can press down OgLuc processed is luminous.This shows that OgLuc variant can carry out MULTIPLE COMPOSITE, Qi Zhong with another luciferase in report measures First report detects the luminescence from OgLuc variant in measuring.
Table 41:PBI-3077 and PBI-1424 is to by utilizing PBI-3939 to express the bacteria lysis of 9B8opt as substrate The luminous effect that thing generates
C. the spectral resolution between OgLuc variant L27V and LUC Photinus pyralis LUC Photinus pyralis FL (Fluc) is analyzed.Will without phenol red+ 0.1%DMEM in the L27V of purification (previously described；9.54pM) with containing 20 μMs of PBI-3939's Measure reagent (previously described) to mix mutually.By the LUC Photinus pyralis LUC Photinus pyralis FL of the purification in same medium (Restructuring luciferase；Promega Corp.；271ng/mL) with detectable (100mM HEPES, PH7.4,1mM CDTA, 16mM MgSO4,1%NP-9 (v/v), 0.1%DF204,5mM ATP, 50mM DTT, 333 μMs of fluoresceins) mix mutually.By 1X Renilla Luciferase Assay Lysis Buffer The sea pansy of the purification in (Promega Corp.) belongs to luciferase (5ng/mL GST-sea pansy genus) and belongs to luciferase survey with sea pansy Determine in buffer 10.5 μMs of natural coelenterazine to mix mutually.Measure the luminescence of L27V and Fluc after 3 minutes and measure after 10 minutes Sea pansy belongs to the luminescence (Figure 71) of luciferase.
D. as another example, the OgLuc variant of the present invention can be used as transcribed reporter and with aequorin or LUC Photinus pyralis LUC Photinus pyralis FL biosensor (or with both the simultaneously) pairing of cAMP circulation exchange is to detect in simple sample Multiple approach, such as, for detecting and/or measure the aequorin of calcium, for detecting and/or measure the biological sense of cAMP Receiver, and for monitoring the OgLuc variant of downstream gene expression.
E. other examples with the OgLuc variant MULTIPLE COMPOSITE of the present invention include:
I) the OgLuc variant containing the present invention and the construct transfectional cell of LUC Photinus pyralis LUC Photinus pyralis FL are utilized.After transfection, can add Adding the first reagent with cell lysis and provides substrate to generate the luminescence of the first luciferase.Then can measure from the first fluorescence The luminescence of element enzyme.Then can add the second reagent from the luminescence of the first luciferase and provides substrate with glimmering from second with cancellation Light element enzyme generates luminescence.Then the luminescence from the second luciferase can be measured.Which kind of luciferase is selected first to measure only Depend on the luminous ability utilizing the second reagent cancellation from the first luciferase.For this example, can first measure from The luminescence of OgLuc variant, owing to showing that the fluorescein (substrate of LUC Photinus pyralis LUC Photinus pyralis FL) of high concentration suppresses OgLuc variant Activity.
Ii) the OgLuc variant containing the present invention and the construct transfectional cell of LUC Photinus pyralis LUC Photinus pyralis FL are utilized.After transfection, can Adding the first reagent, it contains living cells substrate to generate the luminescence of the first luciferase.Then will measure from the first fluorescence The luminescence of element enzyme.Then add the second reagent with cell lysis, cancellation from the first luciferase luminescence and provide substrate with Generate the luminescence from the second luciferase.Then the luminescence from the second luciferase will be measured.This is to i) similar, different It is that cell cracking will limit the use of living cells substrate beneficially cancellation further from the luminescence of the first luciferase.
Iii) the OgLuc variant of the present invention and the construct transfectional cell of LUC Photinus pyralis LUC Photinus pyralis FL are utilized.After transfection, can add Adding a kind of reagent, it contains substrate to generate luminescence from two kinds of luciferases, but from the luminescence of every kind of luciferase at spectrum Upper different.The emission maximum of OgLuc variant is about some substrate of 460nm and LUC Photinus pyralis LUC Photinus pyralis FL, such as, 5 '-chlorine For fluorescein and 5 '-methyl fluorescein, the emission maximum of about 610nm can be produced.Therefore, can from blue emission to red emission Some can be had overlapping, may be non-overlapping from red emission to blue emission, show may seldom relate to or be not related to Mathematical Correction.
Iv) the OgLuc variant containing the present invention and the construct transfectional cell of LUC Photinus pyralis LUC Photinus pyralis FL are utilized.After transfection, can Adding a kind of reagent, it contains living cells substrate to generate luminescence from two kinds of luciferases.Unique feature of this example be At a temperature of living cells measures, such as, 37 DEG C, fire fly luminescence trends towards being changed into redness, therefore, optional the most different glimmering Light element derivant is as the living cells substrate of LUC Photinus pyralis LUC Photinus pyralis FL to generate luminescence, and it is spectrally different from OgLuc variant.
V) the OgLuc variant containing the present invention and sea pansy is utilized to belong to the construct transfectional cell of luciferase.After transfection, can add Adding the first reagent with cell lysis and provides substrate to generate the luminescence of the first luciferase.Then will measure from the first fluorescence The luminescence of element enzyme.Then by add the second reagent with cancellation from the luminescence of the first luciferase and provide substrate with generate from The luminescence of the second luciferase.Then the luminescence from the second luciferase will be measured.Which kind of luciferase is selected first to measure It is only dependent upon the luminous ability utilizing the second reagent cancellation from the first luciferase.For this example, need to use cancellation OgLuc variant or sea pansy belong to the inhibitor that luciferase is luminous.
Vi) the OgLuc variant containing the present invention and the construct transfectional cell of Pleonomus luciferase are utilized.After transfection, can Add a kind of reagent, its containing substrate to generate luminescence from two kinds of luciferases, but from the luminescence of every kind of luciferase at spectrum On be different because Pleonomus luciferase to utilize natural fluoresence element to generate red shift luminous.
Embodiment 47 circulation exchange
Generate two kinds of circulation exchange (CP) form: CP84 and CP95 of L27V variant.Number designation refers to N-terminal residue (such as, " 84 " represent the new N-end of CP form).
For building circulation exchange, utilize non junction (" CP84 non junction " (SEQ ID NO:97 and 98) and " CP95 is without connecing Head " (SEQ ID NO:105 and 106)) or 5 (" CP845AA joint " (SEQ ID NO:99 and 100) and " CP955AA joints " (SEQ ID NO:107 and 108), 10 (" CP8410AA joint " (SEQ ID NO:101 and 102) and " CP9510AA joints " (SEQ ID NO:109 and 110) or 20 (" CP8420AA joint " (SEQ ID NO:103 and 104) and " CP9520AA joints " (SEQ ID NO:111 and 112) Amino acid linker, (GSSGG) n (SEQ ID NO:113) will between N-end and C-end Previous N-end is together with C-terminal fusion.(note: L27V originates in phenylalanine at N-end, i.e. MVF." MV " deposits It is in " non junction " construct, but is not present in " joint " construct).After circulation exchange, CP L27V variant clone is arrived In pF1K carrier.Previously described standard induction scheme of leaving is utilized to utilize CP vector Bacillus coli cells and minimum Limit culture medium is cultivated.For each CP construct, cell is grown in 8 holes of 96 hole flat boards.After induction, mixing comes From 8 holes of each sample, and 10 μ L are cracked in 40 μ L lysis buffers (100mM MES pH6.0,0.3X PLB, 0.3mg/mL lysozyme, 0.003U/ μ L DNA enzymatic I and 0.25%NP-9(v/v)).Then by lysate with 1: 100 (having the CP form of joint) or 1: 1000 (without the form of CP) are diluted in lysis buffer.Do not dilute jointless CP form.(previously described) is diluted with three replication lysates or lysate in 50 μ L measure reagent.As previously Described measurement is luminous (Figure 72).
Embodiment 48-identifies other sites of circulation exchange
For identifying other CP sites, determine that CP site is on the impact of uciferase activity " tethers " of studying between fragment Purposes, generate have at the most every 3rd site (that is, aminoacid) place of L27V variant circulation exchange CP construct (ginseng See Figure 73 E).Skilled persons will understand that other sites, such as, the first site and the second site, it is possible to be examined And utilize the scheme according to manufacturer described herein in circulation exchange OgLuc variant described herein.Such as, Find that L27V variant is especially allowed for circulation exchange, especially in fragment (such as, the impression based on cAMP/RIIbB of exchange Device) between place the situation of relatively large binding structural domain.In each site, with the addition of joint GSSGG-GSSGG-EPTT-ENLYFQS-DN-GSSGG-GSSGG (SEQ ID NO:328).The sequence of underscore refers to TEV protease recognition site.Joint Purpose is to provide sufficiently long tethers between two Variants Fragments thus it can produce the side of functional luciferase Formula is connected.Use TEV protease recognition site to provide the instrument (in the presence of TEV protease) of destruction tethers thus can grind Study carefully its importance maintaining activity.The use of TEV protease recognition site constructs model to predict which CP site will be for Protein complementation measure (PCA) be useful or for biosensor application be useful (such as, between CP site insert should Answer element).
Before TEV cracks, how seen activity represents the performance in tethers state of the two halves of variant enzyme.TEV and knowledge The combination in other site causes cracking, thus is separated by the two halves of variant enzyme.Representative is not induced by the sample utilizing TEV to crack State also provides the expected instruction of how many backgrounds.Activity relatively low after TEV cracking represents that two halves can not be in the feelings without induction Link together under condition.Show after TEV cracking that a large amount of samples lost of activity represent will be in PCA and biosensor application The site worked.In the case of PCA, the two halves of variant enzyme by with can induction binding events after be connected (tethers) Binding partners merge.In the case of biosensor, after the conformational change of zygotic induction occurs, two halves are by " tethers ". One example of PCA is by for making the half of L27V and FRB fusion and second half merge with FKBP.Will when being exposed to rapamycin Make two halves close to (Banaszynski etc., J.Am.Chem.Soc, 127 (13): 4715-4721 (2005)).Biosensor should An example between CP site, insert ring-type AMP binding structural domain (such as, RIIbB) and by ring-type AMP by being Combination with binding structural domain and induced conformational changes.
After generating each CP L27V construct, in Fructus Tritici aestivi, escherichia coli and mammalian cell, express CP enzyme also TEV protease is utilized to digest to study uciferase activity.
1., for be analyzed in Fructus Tritici aestivi, utilize according to the explanation of manufacturerT7Coupled Wheat Germ Extract System (Promega Corp.) expresses CP construct.WillReactant is diluted in 1X with 1: 100 In PBS+0.1% gelatin and 20 μ L are added to TEV reaction reagent (the 5 μ L20X ProTEV buffer (Promega of 25 μ L Corp.), 1 μ L100mM DTT and 2 μ L10U ProTEV Plus (Promega Corp.)).Utilize water by digestion reaction thing Volume increases to 100 μ L, and hatches 60 minutes at 30 DEG C.Also it is prepared for the control sample without TEV protease.Then by 10 The sample of the digestion of μ L adds 40 μ l DMEM to, to the final volume of 50 μ L, and measures (as previously in the mensuration reagent of 50 μ L Described；100μM PBI-3939).Measure luminescence (Figure 73 A-D) as described earlier.
2., for be analyzed in mammalian cell, utilize reverse transfection procedure to utilize CPL27V variant to transfect HEK293 Cell.In short, 1ng CP L27V plasmid DNA is mixed mutually with 1 μ g carrier DNA and add in the hole of 12 hole flat boards thin In born of the same parents.Then by cell at 37 DEG C, 5%CO2Under hatch 16 hours.Then cell is prepared by removal culture medium from cell Lysate, washed cell add 1mL1X PLB in 1X PBS.Then 1: 10 dilution in the 1X PBS containing 0.1% gelatin Lysate.Then as described earlier the lysate of the dilution of 40 μ L is used in TEV protease digestion.Digestion by 10 μ L With mixing without phenol red DMEM of 40 μ L, and the mensuration reagent adding 50 μ L is (previously described；100μM PBI-3939). Measure luminescence (Figure 73 H) as described earlier.
3., for be analyzed in escherichia coli, at 30 DEG C, grow the escherichia coli training overnight expressing CP L27V variant Support.These cultures (be diluted in LB+ antibiotic at 1: 100) are used to generate the new starter culture for final induction.? At 37 DEG C, 2.5 hours (OD of starter culture are hatched under vibration600It is about 0.5).Add rhamnose (final concentration of 0.2%), will Cultivation moves on at 25 DEG C, and hatches 18 hours under vibration.
For generating lysate, by 50 μ L0.5X FASTBREAKTMCell lytic reagent (Promega Corp.) adds to In the culture of the induction of 950 μ L, and mixture is hatched 30 minutes at 22 DEG C.Utilize TEV the most as described earlier The culture of the cracking of protease digestion 50 μ L, and at room temperature hatch 2 hours.
In order to analyze, by lysate 1: 10,000 is diluted inMammalian Purification In Buffer (Promega Corp.) and (as described earlier at the mensuration reagent of 50 μ L；100 μMs of PBI-3939) in right 50 μ L are measured.At 5 minutes points, measure that substrate is luminous and the luminescence (Figure 73 F) of TEV induction as described previously Determination response (Figure 73 G).
Figure 73 A-D shows the base of the different CP-TEV protease L27V construct expressed in wheat malt germ extract The end, is luminous.Figure 73 E summarizes the CP variant (response is that multiple reduces) deriving TEV protease response, shows that CP variant can As TEV sensor, i.e. it can represent " tethers dependency ".Student is utilized to check (not paired p value；Confidence level 0.03) enter The significance of the variant that one step card display at least 1.2 multiple changes.These results represent that each CP variant can generate luminescence.
Different CP-TEV protease L27V constructs is expressed in HEK293 cell.Use previously described reversely turning Dye scheme utilizes the carrier DNA of 1 μ g to transfect 1ng DNA/ hole.The culture medium of the 1mL in 12 hole flat boards is cultivated each Cell sample.By removing culture medium and adding the 1X PLB of 1mL and prepare cell lysate.In 1X PBS+0.1% gelatin 1: 10 dilute sample.Set up the dilute sample of the 40 μ L digested for TEV.As described earlier by the digestion reaction thing of 10 μ L Add in the PBI-3939 without phenol red DMEM and 50 μ L of 40 μ L.Figure 73 H shows expression in HEK293 cell The luminescence of different CP-TEV protease L27V constructs.
Data in Figure 73 A-H show L27V variant can in the circulation exchange of different sites, and different sites have with The different dependency that tethers length is relevant.Mammalian cell data and Fructus Tritici aestivi data show and utilize TEV to crack Similar multiple reduces.Rely more on tethers, i.e. TEV protease is cracked more sensitive CP L27V variant is the potential of PCA Material standed for.The CP L27V variant being relatively independent of tethers is probably the potential material standed for measured from complementation/dimerization.
Embodiment 49 protein complementation measures
Protein complementation measures the instrument that (PCA) provides the interaction of two i.e. polypeptide of biomolecule of detection.PCA utilizes Two fragments of identical albumen such as enzyme, when said two fragment being reconstituted for functional close to Shi Qike mutually Activity albumen.In the site that tolerance separates, the OgLuc variant of the present invention can be divided into two fragments.Then, separation Each fragment of OgLuc variant can with think interactive a pair polypeptide interested (such as, FKBP and FRB) wherein it One merges.If two peptide species interested the most in fact interact, then OgLuc fragment mutually close to and reconstitute The OgLuc variant of functional activity.Then the novel coelenterazine utilizing natural or known coelenterazine or the present invention can be examined Survey and measure the activity of the OgLuc variant reconstituted.In another example, montage OgLuc variant can be used for and lac-Z (Langley etc., PNAS, 72:1254-1257 (1975)) or ribonuclease S (Levit and Berger, J.Biol.Chem., 251:1333-1339 (1976)) in similar more general complementary system.Especially, can be by known and another OgLuc variant sheet OgLuc Variants Fragments (named " A ") and target protein that section (" B ") is complementary merge, and are split by the cell containing fragment B or cell Solve the luminescence in thing and monitor the fusant of gained.The source of fragment B can be that identical cell is (in chromosome or at another On plasmid), or its lysate that can be derived from another cell or the albumen of purification.Utilize fragment B and polypeptide all if It is attached to solid carrierBetween fusion can catch or fix this identical fusion protein (fragment A).So Rear available luminescence proves the amount of the material successfully catching or quantitatively being caught.For protein complementation according to manufacturer Scheme can be carried out according to U.S.'s published application the 2005/0153310th, and it is incorporated herein by.
1. the following 9B8opt PCA construct that generates:
-p9B8PCA1/4=pF5A/Met-[9B8opt (51-169)]-GGGGSGGGSS-FRB (SEQ ID NO:331 and 332) &pF5A/FKBP-GGGSSGGGSG-[9B8opt (1-50)] (SEQ ID NO:337 and 338)
-p9B8PCA1/2=pF5A/Met-[9B8opt (51-169)]-GGGGSGGGSS-FRB (SEQ ID NO:331 and 332) &pF5A/ [9B8opt (1-50)]-GGGGSGGGSS-FRB (SEQ ID NO:333 and 334)
-p9B8PCA2/3=pF5A/ [9B8opt (1-50)]-GGGGSGGGSS-FRB (SEQ ID NO:333 and 334) & PF5A/FKBP-GGGSSGGGSG-[9B8opt (51-169)] (SEQ ID NO:335 and 336)
-p9B8PCA3/4=pF5A/FKBP-GGGSSGGGSG-[9B8opt (51-169)] (SEQ ID NO:335 and 336) &pF5A/FKBP-GGGSSGGGSG-[9B8opt (1-50)] (SEQ ID NO:337 and 338)
Explanation according to manufacturer utilizesPCA construct is transfected into the HEK293 in 96 hole flat boards by HD In cell (15,000 cells/well).Then at 37 DEG C, 5%CO2Lower night incubation cell.After transfection, by the cultivation on cell Base is with being independent of CO containing 10%FBS2Culture medium substitute.Then the mensuration reagent containing 20 μMs of PBI-3939, and 28 are added On Varioskan Flash, luminescence is measured at DEG C.Then 100 μMs of rapamycins are added in hole, and test constantly luminescence 1 Hour.By calculating multiple response with all luminescences in given hole divided by the luminescence of the rapamycin pretreatment in identical hole (Figure 74).
2. for proving OgLuc variant purposes in PCA, L27V02A Variants Fragments is complementary with FKBP or FRB, and survey Interaction between amount FKBP and FRB.
Table 42 lists different protein complementations (PCA) construct generating and checking." 2/3 " represents variant and recruits mutually Right, wherein 1) " old " C-end (the original C-terminal of " old "=L27V02A) of L27V02A is the C-end companion of FKBP；With 2) " old " N-end of L27V02A is the N-end companion of FRB." 1/4 " represents variant pairing, wherein 1) " old " N-of L27V02A End is the C-end companion of FKBP；With 2) " old " C-end of L27V02A is the N-end companion of FRB.For all structures Body, FKBP is positioned at the N-end of L27V02A fragment, and FRB is positioned at the C-end of L27V02A fragment.Such as, generate 157 (seeing table 42, " 2/3 " and " 1/4 " #s11 and 12 (SEQ ID NO:288-295)), the 103rd (see table 42, " 2/ 3 " and " 1/4 " #s9 and 10 (SEQ ID NO:296-303)) and the 84th (see table 42, " 2/3 " and " 1/4 " #s7 and 8 (SEQ ID NO:304-315)) there is the PCA construct of splice site.Generate other PCA constructs (SEQ ID NO:343-426 And 428-440) (seeing table 21).
Table 42
Table 21
PCA construct
SEQ ID NO:343 (pCA31pCA L27V02A45-169FRB)
SEQ ID NO:344 (pCA31pCA L27V02A45-169FRB)
SEQ ID NO:345 (pCA32FKBP L27V02A1-44)
SEQ ID NO:346 (pCA32FKBP L27V02A1-44)
SEQ ID NO:347 (pCA33pCA L27V02A46-169FRB)
SEQ ID NO:348 (pCA33pCA L27V02A46-169FRB)
SEQ ID NO:349 (pCA34pCA FKBP1-45L27V02A)
SEQ ID NO:350 (pCA34pCA FKBP1-45L27V02A)
SEQ ID NO:351 (pCA35pCA L27V02A100-169FRB)
SEQ ID NO:352 (pCA35pCA L27V02A100-169FRB)
SEQ ID NO:353 (pCA36FKBP L27V02A1-99)
SEQ ID NO:354 (pCA36FKBP L27V02A1-99)
SEQ ID NO:355 (pCA37L27V02A101-169FRB)
SEQ ID NO:356 (pCA37L27V02A101-169FRB)
SEQ ID NO:357 (pCA38FKBP1-100L27V02A)
SEQ ID NO:358 (pCA38FKBP1-100L27V02A)
SEQ ID NO:359 (pCA39L27V02A102-169FRB)
SEQ ID NO:360 (pCA39L27V02A102-169FRB)
SEQ ID NO:361 (pCA40FKBP L27V02A1-101)
SEQ ID NO:362 (pCA40FKBP L27V02A1-101)
SEQ ID NO:363 (pCA41L27V02A143-169FRB)
SEQ ID NO:364 (pCA41L27V02A143-169FRB)
SEQ ID NO:365 (pCA42FKBP1-142L27V02A)
SEQ ID NO:366 (pCA42FKBP1-142L27V02A)
SEQ ID NO:367 (pCA43L27V02A145-169FRB)
SEQ ID NO:368 (pCA43L27V02A145-169FRB)
SEQ ID NO:369 (pCA44FKBP1-144L27V02A)
SEQ ID NO:370 (pCA44FKBP1-144L27V02A)
SEQ ID NO:371 (pCA45L27V02A147-169FRB)
SEQ ID NO:372 (pCA45L27V02A147-169FRB)
SEQ ID NO:373 (pCA46FKBP-L27V02A1-146)
SEQ ID NO:374 (pCA46L27V02A-FKBP1-146)
SEQ ID NO:375 (pCA47L27V02A148-169FRB)
SEQ ID NO:376 (pCA47L27V02A148-169FRB)
SEQ ID NO:377 (pCA48FKBP-L27V02A1-147)
SEQ ID NO:378 (pCA48FKBP-L27V02A1-147)
SEQ ID NO:379 (pCA49L27V02A156-169FRB)
SEQ ID NO:380 (pCA49L27V02A156-169FRB)
SEQ ID NO:381 (pCA50FKBP-L27V02A1-155)
SEQ ID NO:382 (pCA50FKBP-L27V02A1-155)
SEQ ID NO:383 (pCA51L27V02A158-169FRB)
SEQ ID NO:384 (pCA51L27V02A158-169FRB)
SEQ ID NO:385 (pCA52FKBP1-157L27V02A)
SEQ ID NO:386 (pCA52FKBP1-157L27V02A)
SEQ ID NO:387 (pCA53L27V02A166-169FRB)
SEQ ID NO:388 (pCA53L27V02A166-169FRB)
SEQ ID NO:389 (pCA54FKBP L27V02A1-165)
SEQ ID NO:390 (pCA54FKBP L27V02A1-165)
SEQ ID NO:391 (pCA55FKBP L27V02A1-47)
SEQ ID NO:392 (pCA55FKBP L27V02A1-47)
SEQ ID NO:393 (pCA56L27V02A48-169-FRB)
SEQ ID NO:394 (pCA56L27V02A48-169-FRB)
SEQ ID NO:395 (pCA57FKBP L27V02A1-49)
SEQ ID NO:396 (pCA57FKBP L27V02A1-49)
SEQ ID NO:397 (pCA58pCA L27V02A50-169FRB)
SEQ ID NO:398 (pCA58pCA L27V02A50-169FRB)
SEQ ID NO:399 (pCA59FKBP L27V02A1-82)
SEQ ID NO:400 (pCA59FKBP L27V02A1-82)
SEQ ID NO:401 (pCA60L27V02A83-169-FRB)
SEQ ID NO:402 (pCA60L27V02A83-16-FRB)
SEQ ID NO:403 (pCA61FKBP L27V02A1-84)
SEQ ID NO:404 (pCA61FKBP L27V02A1-84)
SEQ ID NO:405 (pCA62L27V02A85-169-FRB)
SEQ ID NO:406 (pCA62L27V02A85-169-FRB)
SEQ ID NO:407 (pCA63FKBP L27V02A1-122)
SEQ ID NO:408 (pCA63FKBP L27V02A1-122)
SEQ ID NO:409 (pCA64L27V02A123-169-FRB)
SEQ ID NO:410 (pCA64L27V02A123-169-FRB)
SEQ ID NO:411 (pCA65FKBP L27V02A1-123)
SEQ ID NO:412 (pCA65FKBP L27V02A1-123)
SEQ ID NO:413 (pCA66L27V02A124-169FRB)
SEQ ID NO:414 (pCA66L27V02A124-169FRB)
SEQ ID NO:415 (pCA67L27V02A1-168)
SEQ ID NO:416 (pCA67L27V02A1-168)
SEQ ID NO:417 (pCA67L27V02A1-168)
SEQ ID NO:418 (* pCA68L27V02A169FRB)
SEQ ID NO:419 (* pCA68L27V02A169FRB)
SEQ ID NO:420 (pCA69FKBP L27V02A1-166)
SEQ ID NO:421 (pCA69FKBP L27V02A1-166)
SEQ ID NO:422 (* pCA70L27V02A167-169FRB)
SEQ ID NO:423 (* pCA70L27V02A167-169FRB)
SEQ ID NO:424 (pCA71FKBP L27V02A1-164)
SEQ ID NO:425 (pCA71FKBP L27V02A1-164)
SEQ ID NO:426 (pCA72L27V02A165-169FRB)
SEQ ID NO:428 (pCA72L27V02A165-169FRB)
SEQ ID NO:429 (pCA73FKBP L27V02A1-162)
SEQ ID NO:430 (pCA73FKBP L27V02A1-162)
SEQ ID NO:431 (pCA74L27V02A163-169FRB)
SEQ ID NO:432 (pCA74L27V02A163-169FRB)
SEQ ID NO:433 (pCA75FKBP L27V02A1-160)
SEQ ID NO:434 (pCA75FKBP L27V02A1-160)
SEQ ID NO:435 (pCA76L27V02A161-169FRB)
SEQ ID NO:436 (pCA76L27V02A161-169FRB)
SEQ ID NO:437 (pCA77FKBP L27V02A1-158)
SEQ ID NO:438 (pCA77FKBP L27V02A1-158)
SEQ ID NO:439 (pCA78L27V02A159-169FRB)
SEQ ID NO:440 (pCA78L27V02A159-169FRB)
Complementary pair described in table 42 is cloned in pF4Ag carrier as described earlier.Then knit red at rabbit net Cell lysate (RRL；Promega Corp.) or wheat malt germ extract (Promega Corp.) middle expression PCA construct (900μL).By about the expression response thing of 1.25 μ L of each PCA pair and the 2X of 10 μ L combine buffer (100mM HEPES, 200mM NaCl, 0.2%CHAPS, 2mM EDTA, 20% glycerol, 20mM DTT, pH7.5) and the mixing of 7.5 μ L aqueous phases, and by 18 μ L transfers in the hole of 96 hole flat boards.Add the rapamycin (final concentration of 0.5 μM) of 5 μMs of 2 μ L and at room temperature hatch 10 Minute.
After hatching, add the PBI-3939 (50X stock solution measure in buffer be diluted to 1X) at room temperature of 100 μ L Hatch 3 minutes.Measure luminescence (Figure 76 A-B: Fructus Tritici aestivi as described earlier；Figure 76 C-D: rabbit reticulocyte；Figure 76 E- F: cell free system [which kind of system？WG or RRL？]；Figure 76 G:HEK293 cell).
Figure 76 A-G shows the luminescence of different protein complementations (PCA) L27V pair: utilize 2/3 configuration (figure as described 76A and 76C) or 1/4 configuration (Figure 76 B and 76D) the L27V fragment of every pair is merged with FKBP or FRB, with at wheat germ FKBP and FRB monitored in bud extract (Figure 76 A and 76B) and rabbit reticulocyte lysate (RRL) (Figure 76 C and 76D) Interaction；Luminescence (Figure 76 E) with different protein complementation (PCA) negative controls.Measure at cell free system (RRL) (Figure 76 F) and HEK293 cell (Figure 76 G) utilize the luminescence of the different protein complementation L27V of 1/4 configuration.In Figure 76 A-G Tables of data understands that multiple different disappearance (that is, the small fragment of L27V variant) is functional.
3., for show the PCA construct purposes to PCA based on cell, construct is transfected in HEK293 cell and profit It is measured with PBI-4377.Will be from each PCA to the plasmid DNA (5ng) of (6,12,55,84 and 103) and 40ng carrier DNA (pGEM-3fz) and 5 μ LMix mutually and at room temperature hatch 5 minutes.Then add HD (0.15 μ L) also the most at room temperature hatches 15 minutes.DNA transfection mixture is added to the DMEM (nonreactive containing 10%FBS Raw element) 100 μ L HEK293 cell (1.5x105Individual cell/mL) in, and transfer in the hole of 96 hole flat boards, and at 37 DEG C, 5%CO2Lower overnight incubation.
After transfection, remove culture medium and with containing 20 μMs or 50X PBI-4377 be independent of CO2Culture medium substitute also Without CO2Hatch at 37 DEG C 2 hours in the case of regulation.Measure luminescence, add 10 μ L rapamycins, and every 2 minutes again Measure luminescence, continue 2 hours (Figure 76 A-C).
4. for proving that PCA construct, for identifying the purposes of the inhibitor of protein-protein interaction, uses this embodiment #2 described in construct.
As described earlier by the complementary pair 103 " 2/3 ", 157 " 2/3 " described in table 42,103 " 1/4 " and 157 " 1/4 " is cloned in pF4Ag carrier.Then in rabbit reticulocyte lysate, express PCA according to the explanation of manufacturer to build Body (25 μ L) (RRL；Promega Corp.).The expression response thing of the 1.25 μ L about every kind of PCA and the 2X of 10 μ L are combined slow Rush liquid (100mM HEPES, 200mM NaCl, 0.2%CHAPS, 2mM EDTA, 20% glycerol, 20mM DTT, pH7.5) and 7.5 μ L aqueous phase mixes, and is transferred to by 16.2 μ L in the hole of 96 hole flat boards.The FK506 utilizing different amounts checks rapamycin.To This reaction is added FRB-FKBP binding inhibitors, FK506 (10X), and at room temperature incubation reaction 10 minutes.Add 15nM thunder Handkerchief mycin (10X stock solution) is to obtain the final concentration of 1.5nM rapamycin and at room temperature to hatch 2 hours.After hatching, add The PBI-3939 of 100 μ L (50X stock solution is diluted to 1X in measuring buffer) also at room temperature hatches 3 minutes.?Luminescence is measured on photometer.Figure 77 demonstrates PCA construct disclosed herein and can be used for identifying albumen-egg The white inhibitor interacted.
5. for proving the purposes of the PCA construct of cracking form, by complementary pair 103 " 2/3 ", 157 " 2/3 " and 103 " 1/4 " It is transfected in HEK293 cell and utilizes PBI-3939 to be measured.By from the 0.5ng plasmid of each PCA pair and 5 μ LMix mutually with 49ng pGEM-3zf (Promega Corp.).Sample mixture is at room temperature hatched 5 Minute.Then by 0.15 μ LHD adds in sample mixture and at room temperature hatches 15 minutes.By 100 μ L HEK293 cell in DMEM containing 10%FBS is with 1.5x105Individual cell/mL adds in each sample mixture.Then will Cell sample is transferred in the hole of 96 hole flat boards and at 37 DEG C, 5%CO2Lower overnight incubation.
Second day, 10 μMs of rapamycins (final concentration 1 μM) of 11.1 μ L are added in the hole of half and by 11.1 μ L water Add in second half hole.96 hole flat board is hatched 1 hour at 37 DEG C.By the mensuration reagent+PBI-3939 of 100 μ L (previously Described, 2 μ L50X PBI-3939 and 98 μ L measure reagent and mix mutually) add each hole to and at 37 DEG C, hatch flat board 4 Minute.At 37 DEG C,Luminescence is measured with the 0.5s time of integration and 1 reading on photometer.(Figure 76 H).
Embodiment 50 OgLuc cAMP biosensor
Not only by concentration, also by the regulation of enzymatic activity, the OgLuc variant of the present invention can be connected with light output. Such as, by the cAMP-binding structural domain from protein kinase A is incorporated into circulation exchange OgLuc variant in and develop CAMP biosensor.Can be by by method as known in the art (such as, U.S. Published Application the 2005/0153310th) The OgLuc variant of the present invention is circulated exchange in the site of tolerance exchange.The OgLuc variants chimeric egg of the circulation exchange of gained In vain when expressing in mammalian cell can as the intracellular biological sensor of cAMP functionating.CAMP and biological sense After receiver combines, biosensor experienced by conformational change, creates the luciferase of activity.Use adenyl cyclase forskolin Process cell, the luminous increase of the concentration of the increase with forskolin should be caused.Target includes but not limited to calcium (Ca+2), cGMP Can be by by suitable combination with the similar biosensor of protease such as caspase with marmor erodens (TEV) The cracking site of domain or every kind is incorporated into the OgLuc variant of circulation exchange and develops.
Answering of the OgLuc as biosensor is demonstrated by analyzing the variant 9B8opt in cAMP sensor environment With.Generate the construct of the circulation exchange of RII β B subunit containing the protein kinase A that flank is OgLuc variant sequence thereof, and such as Expressing in cell free system described in PCT application PCT/US2007/008176, selects the most as described below Select the site for circulating exchange.Nascent protein is measured when there is and do not exist cAMP.By activity (+) cAMP/ (-) cAMP's Ratio determines the response to cAMP.
Based on previously described in PCT/US2010/33449, with some fatty acid binding protein of known structure Similarity, has constructed the structural model of OgLuc.The orderly sequence of model prediction standard protein structural motif；Alpha-helix and β-lamella.Have selected the region of transition between these structural details and exchange site (seeing table 43) as circulation.
1. the template being used for expressing biosensor construct consists of: in plasmid pF5 (Promega Corp.) C-end OgLuc sequence-RII β B sequence-N-end OgLuc sequence.T7Coupled Wheat Germ Extract System (Promega Part#L4140) is used for translating this construct.Wheat Germ Extract Reaction Containing 25 μ LWheat Germ Extract (L411A), 2 μ LReaction Buffer (L462A), 1 μ L Amino Acid Mixture, Complete (L446A), 1 μ L(40U/ μ L) (N2615), 1 μ LT7RNA Polymerase (L516A), 1.0 μ g DNA profilings and make cumulative volume increase to 50 μ L with the water of nuclease free.Hatch anti-at 30 DEG C Answer mixture 120 minutes.
By adding the 50 μ L OgLuc Glo with or without 100 μMs of cAMP in 50 μ L OgLuc translation mixture Reagent (100mM MES (pH6.0), 1mM CDTA, 150mM KCl, 35mM thiourea, 2mM DTT, 0.25%NP-9 (v/v), 0.025%DF204 and 20 μMs of PBI-3939) carry out OgLuc activity survey Fixed, the action mechanics reading 30 minutes of going forward side by side (F500 flat bed reader).By with by containing The luminescence that the biosensor of cAMP generates determines response (table divided by the luminescence generated by the biosensor without cAMP 43)。
Table 43: the OgLuc biosensor of the circulation exchange response to cAMP
CP site Response
27 2.6X
51 2.2X
84 1.5X
122 4.3X
147 1.9X
157 5.6X
2. as described in 1, at CP site 51, build the cAMP biosensor of the 9B8opt of circulation exchange.Then root Utilize according to the explanation of manufacturerHD biosensor is transfected in the HEK293 cell in 96 orifice plates (15, 000 cells/well) in, and at 37 DEG C, 5%CO2Lower night incubation.After transfection, remove culture medium and with containing 10%FBS not Rely on CO2Culture medium substitute.Then at 37 DEG C, 5%CO2Lower incubated cell 2 hours, adds the FSK of variable concentrations afterwards. The most again at 37 DEG C, 5%CO2Lower incubated cell 3 hours.Then add 6 μMs of PBI-3939, after 13 minutes, measure luminous (figure 78)。
3. by (" CP " of circulation exchange；Such as CP6 refers to that new the 1st that old the 6th residue becomes after methionine is residual Base) and (" SS " of direct montage；Such as, SS6 refers to the sensor positioned as follows: OgLuc (1-6)-RII β b binding site (SEQ ID NO:441 and 442)-OgLuc (7-169)) form L27V be used as cAMP biosensor (SEQ ID NO:467-574). The CP (SEQ ID NO:467-498 and 555-574) and SS (SEQ ID NO:499-of derivative L27V variant as described earlier 554) form and according to the explanation of manufacturer at rabbit reticulocyte lysate (RRL；Promega Corp.) middle expression.RIIβb Joint sequence between C-end and the OgLuc luciferase sequence of binding site is GGGTCAGGTGGATCTGGAGGTAGCTCTTCT (SEQ ID NO:575).N-end and the OgLuc of RII β b binding site are glimmering Joint sequence between light element enzyme sequence is that AGCTCAAGCGGAGGTTCAGGCGGTTCCGGA (SEQ ID NO:576) is by 3.75 Expression response thing and the 1.25 μ L4X cAMP (final concentration 1nM-0.1mM) of μ L mix mutually and at room temperature hatch 15 minutes.Hatch After, add the PBI-3939 (50X stock solution is diluted to 1X in measuring buffer) of 100 μ L and at room temperature hatch 3 minutes.?Measure luminous (Figure 79 A-B) on photometer.Also measure and express and as previously retouched in HEK293 cell The luminescence (Figure 79 C-D) of CP and the SS form of the L27V variant with forskolin process stated.Figure 79 A-D shows disclosed herein Circulation exchanging form and the direct splicing form of OgLuc variant can be used as biosensor.
Embodiment 51 subcellular proteomics and location
For analyzing subcellular proteomics, by U2OS cell with 2x104Individual cell/cm2Be inoculated in culture vessel with glass bottom contains In McCoy ' the s5A culture medium of 10%FBS (Invitrogen).Then at 37 DEG C, 5%CO2Lower incubated cell 24 hours.Then Utilize 1/20 volume transfection mixture (HD and be cloned into the pF5A containing CMV promoter (Promega Corp.) PF5A-CMV-L27V (L27V variant (SEQ ID NO:88)) in carrier or pGEM3ZF (Promega Corp.；Negative right According to)) transfectional cell, and at 37 DEG C, 5%CO2Under hatch 24 hours.After hatching, with containing 0.5%FBS and 100 μMs of PBI-4378 Be independent of CO2Culture medium substitute cell culture medium.After hatching 30 minutes at 37 DEG C, biological at Olympus LV200 Utilize 60X eyepiece to catch unfiltered image (Figure 80 A-B) on illuminating microscope, continue 25,100,1000 and 5000ms.
For analyzing Subcellular Localization, GSSG joint (SEQ ID NO:457 and 458) is utilized to generate containing having IL-6 secretion sequence Row (SEQ ID NO:461 and 462) or GPCR AT1R (the hypertensin 1 type of transcription factor Nrf2 (SEQ ID NO:317) Receptor (SEQ ID NO:459 and 460)) N-end L27V fusant, and be transfected into as described earlier in U2OS cell (Figure 81 A-C).Figure 81 C (" GPRC ") shows the expression of construct, wherein upper at L27V variant sequence thereof of IL6 signal sequence Trip, and AT1R is in the downstream of L27V variant sequence thereof.Also transfect single L27V variant (" fusion ").At 37 DEG C, 5% CO2Under hatch 24 hours after, with being independent of CO containing 0.5%FBS2Culture medium substitute cell culture medium and at 37 DEG C non- CO2The atmosphere of regulation balances 1 hour.Then+200 μMs of PBI-3939 of isopyknic culture medium are added, immediately at Olympus 60X or 150X eyepiece is utilized to catch unfiltered image (Figure 81 A-C) on LV200 bioluminescence microscope.Independent for expressing The cell of L27V, PBI-3939 washed from cell and catch image immediately.
Embodiment 52 monitors intracellular signaling pathway
This embodiment offers two of novel fluorescence element enzyme for monitoring intracellular signaling pathway on protein level Example (is contrasted with the response element example representing transcriptional activation).By variant 9B8opt (SEQ ID NO:24) and IkB (Gross etc., Nature Methods2 (8): 607-614 (2005)) merge (at C-end, i.e. N-IkB-(9B8opt)-C)) Or with ODD (the degrading texture territory (Moroz etc., PLoS One4 (4): e5077 (2009)) of the oxygen dependence of Hif-1-α merge ( N-end, i.e. N-(9B8opt)-ODD-C)).Known with TNF α stimulate after IKB degrading in cell；Therefore, IKB- (9B8opt) construct can be used as living cells TNF α sensor.Known ODD (Hif-1-after stimulating with the compound of inducing hypoxia α) accumulate in cell；Therefore ODD-(9B8opt) can be used as living cells anoxia sensor.
It is as described earlier that by reverse transfection, (5ng (IkB) or 0.05ng (ODD) DNA (mixes with carrier DNA to obtain To 50ng altogether)) in HEK293 cell, express the construct of the fusant containing IkB or ODD and 9B8opt (pF5A) and 37 DEG C, hatch 24 hours under 5%CO2.After transfection, it is independent of CO with fresh containing 0.5%FBS and 20 μMs of PBI-43772Training Support base substitutive medium and make its at 37 DEG C, CO2Balance 4 hours under atmosphere.Then stimulus object is exposed cells to: TNF α is used for IkB fusant express cell and phenanthroline are for ODD fusant express cell.Add DMSO (carrier) to compared with control cells. For TNF α/IKB sample, within 15 minutes, add 100 μ g/mL cycloheximide to prevent new albumen in the precontract adding stimulus object Synthesis.After the time point specified processes, measure the luminescence of cell.For data normalization, each sample that preset time is put The RLU of product is divided by the RLU from the most post-stimulatory same sample.It is then determined that multiple response (Figure 82 A-of each sensor C)。
B. use L27V to come on protein level and monitor oxidative stress signal pathway.L27V or sea pansy are belonged to luciferase (Rluc) merge (at C-end with the Nrf2/NFE2L2 in pF5K expression vector；That is, N-Nrf2-(L27V)-C or N-Nrf2- (Rluc)-C).Keap1 is negative regulation of Nrf2 (SEQ ID NO:217).For verily representing Nrf2-L27V02 albumen The regulation and control of level, coexpression Keap1 is to maintain Nrf2 low-level (passing through ubiquitination).
Expressed in HEK293 cell by transfectional cell while of as described earlier in being inoculated into 96 hole flat boards Nrf2-L27V or Nrf2-Rluc (5ng, pF5K) andFusion protein (pFN21-HT7-Keap1 (SEQ ID NO:316)；50ng), and at 37 DEG C, 5%CO2Under hatch 24 hours.After transfection, with containing 0.5%FBS and 20 μMs PBI-4377 (for L27V) or 20 μMs of ENDURENTM(Promega Corp.) (luciferase is belonged to for sea pansy) fresh It is independent of CO2Culture medium substitutive medium, and at CO at 37 DEG C2Statocyte 4 hours in atmosphere.For carrying out power credit Analysis, uses 20 μMs of D, L sulforaphen or carrier (DMSO).In Figure 83 A, as described earlier at the time point specified Luminescence is measured after reason.For carrying out data normalization, the luminescence of each sample put preset time is divided by after immediately stimulation The luminescence (Figure 83 B-C) of same sample.
C. carry out the Nrf2 sensor described in B and Nrf2 (ARE)-Luc2P reports answering of son (Promega Corp.) The contrast answered.Such as previous screening Nrf2 sensor described in above chapters and sections B and report.For Lampyridea (Luc2P) Report sub-genetic testing, use ONE-GLOTMMeasure reagent.Figure 84 A-B provides the standardization response that Nrf2-L27V was at 2 hours With Nrf2 (ARE)-Luc2P the standardization response of 16 hours.
Embodiment 53 OgLuc variant reports the assessment of son as the bioluminescence utilizing BRET
Bioluminescence Resonance Energy transfer (BRET) allows monitoring protein-protein interaction.At IV and HT7 fusion partner Between check intramolecular energy, wherein HT7 is the most marked fluorogen, i.e. TMR (excitation/emission (excitation/emission) Wavelength=555/585nm) or rhodamine 110 (excitation/emission wavelength=502/527nm).To melt containing the IV-HT7 of embodiment 34 50 μ L bacteria cell cracking things of hop protein and 0.001-10 μM of fluorogen part or at room temperature hatch without part 1 hour.Incubate After educating, by the RENILLA-GLO of the 50 μ L containing 22 μMs of coelenterazine-hTMAdd in the enzyme-ligand mixture of 50 μ L, and at 5 points During clock, wavelength launched in record.Show the IV-HT7 with TMR (Figure 83 A) or rhodamine 110 (" Rhod110 ") (Figure 85 B) The wavelength of example, shows that BRET is bigger, i.e. utilize sieve when the excitation/emission of part is close to the 460nm luminescence peak of OgLuc Red bright 110 bigger.The data show the intramolecular energy on fusion protein can occur OgLuc variant and fluorogen it Between.Three kinds of different comparisons are used to be used for comparing (data are not shown): 1) without HT fusant, 2) HT-of unused HT ligand-labeled Fusant, and 3) (it indicates and relates to for the HT-fusant of labelling of TEV site protein cleavage between OgLuc and HT Close/distance).In three kinds of different comparisons, do not observe BRET, show that HT participates in realizing BRET.Compared with N-end HT7 Relatively for C1+A4E and the IV with C-end HT7, BRET is bigger.
The albumen of embodiment 54 living cells or cracking form is close to measuring
In one example, (CP) of circulation exchange or (SS) OgLuc fusant albumen of directly montage are applied to egg The most close measurement.Exchanged by insertion protease substrate amino acid sequence (such as, TEV) or montage OgLuc is low to generate Bioluminescence.Inactive luciferase by tethers (such as, via gene fusion) in monitoring albumen.Possible interaction Albumen by tethers (such as, via gene fusion) in protease (such as, TEV).When two are monitored protein-interactings or sentence (such as, interacting via composing type, medicine irritation or approach response) time sufficiently close together, luciferase is cleaved to generate increasing High bioluminescent activity.This example can be applicable to the measurement that the albumen in cell or in biochemical measurement is close.Further, The high thermal stability of OgLuc variant luciferase may measure antibody-anti-in the cell of cracking or biochemical measurement Former interaction.
Embodiment 55 bioluminescence assay
1., for determining OgLuc variant purposes in the bioluminescence assay of detection caspase-3 mRNA, utilization includes 9B8opt variant is used in bioluminescence assay by the pre-coelenterazine substrate of DEVD caspase-3 mRNA cleavage sequence.By purification Caspase-3 mRNA mixes mutually with the E. coli lysate sample expressing variant 9B8opt, described E. coli lysate sample Preparing as described in embodiment 27, and 10 times be diluted in containing 100mM MES pH6.0,1mM CDTA, 150mM KCl, 35mM thiourea, 2mM DTT, 0.25%NP-9 (v/v), 0.025%DF204, has or not Do not have in the buffer of the 23.5 μMs of z-DEVD-coelenterazine-h being contained in 100mM HEPES pH7.5.At room temperature utilize Caspase-3 mRNA is hatched 3 hours by lysate sample, and at different time points at Turner MODULUSTMExamine on photometer Survey luminescence.To only contain the sample of bacterial lysate and only contain the sample of caspase-3 mRNA with comparing.Use three repetitions.Figure 86 and table 44 prove 9B8opt and and then other OgLuc variants of the present invention available before-coelenterazine substrate is for bioluminescence To detect enzyme interested in mensuration.
Table 44: hatch together with the caspase-3 mRNA of purification as substrate from utilizing z-DEVD-coelenterazine-h or differ Act the average luminescence in terms of RLU generated in the bacterial lysate of the expression 9B8opt hatched.
Time (minute) Without caspase (RLU) + caspase (RLU)
5 26,023 25,411
15.3 7,707 36,906
29.9 4,013 41,854
60.9 2,305 43,370
190.3 1,155 42,448
2. utilize and include that L27V variant is used for biological sending out by the pre-coelenterazine substrate of DEVD caspase-3 mRNA cleavage sequence In light measurement.By the caspase-3 mRNA (1mg/mL) of purification in 100mM MES pH6 (50 μ L) and mensuration buffer (50 μ L) In 227nM L27V02 variant and 47 μMs of PBI-3741 (z-DEVD-coelenterazine-h) mix mutually.At room temperature incubation reaction 3 Hour, and detect luminescence as described earlier.The mensuration of L27V variant and the mensuration of LUC Photinus pyralis LUC Photinus pyralis FL form will be utilized Compare,3/7-Assay system(Caspase-Glo；Promega Corp.).Table 45 shows L27V variant, and thus other the OgLuc variant of the present invention may utilize pre-coelenterazine substrate in bioluminescence assay with Detect enzyme interested.
Table 45
Embodiment 56 immunoassay
The OgLuc variant of the present invention can be dissolved in multiple different immunoassay concept.Such as, OgLuc variant is What anti-with one or two anti-gene fusion or chemistry was conjugated thinks specific analyte offer detection method.As another example Son, OgLuc variant is to merge with protein A, Protein G, any other peptide of albumen L or known combination Ig fragment or protein gene Or chemistry is conjugated, and then this can be used for the specific antibody that detection is combined with specific analyte.As another example, OgLuc variant is spy that is conjugated with streptavidin gene fusion or chemistry and that be combined with specific analyte for detection Fixed biotinylated antibody.As another example, the complementary fragment of OgLuc variant resists and two anti-gene fusion with one Or chemistry is conjugated, the analyte that wherein an anti-identification is specifically fixed, and two anti-identifications one are anti-, are the form of ELISA sample. OgLuc Variant Activity, i.e. luminous, reconstitutes and is used as the instrument of quantifying analytes in the presence of fixing analyte.
As another example, the complementary pairing of OgLuc variant can be with two Antibody Fusion, and one of them antibody is at one The analyte at specific analyte, and the two separate epi-positions of anti-identification is identified at epi-position.OgLuc Variant Activity will be in analyte In the presence of reconstitute.Method is by quantitative in compliance with the analyte in complex environment such as cell lysate or cell culture medium Measure.As another example, the complementary fragment of OgLuc variant will be with two Antibody Fusion, and one of them antibody recognition is specific Analyte, no matter whether modify, and the two anti-analytes (such as, after post translational modification) only identifying modification.OgLuc variant is lived Property will only reconstitute in the presence of its adorned analyte.Method will be in compliance with complex environment such as cell lysate In the measurement of analyte of modification.As another example, OgLuc variant will be conjugated with analyte (such as, prostaglandin) And use with competitive sandwich ELISA form.
Embodiment 57 dimerization measures
This example prove total length circulation exchange OgLuc variant can merge with corresponding binding partners, such as, FRB and FKBP, and measure for protein complementation type.Between scheme according to manufacturer disclosed herein and traditional protein complementation Key difference be not exist complementation, but there is the dimerization of the enzyme (such as, the OgLuc variant of circulation exchange) of two total lengths Change.
In short, simply configure both of albumen report exchanged for SA circulation and fusion protein companion Merge (seeing Figure 87 A).Such as, each fusion partner can be connected with report mutually isostructural, exchange.Fusion partner Interact report making exchange close to, thus allow to have reconstituting of higher active hybridization report.Newly Type hybridization report includes the part of report of each circulation exchange in the way of reducing structure restriction.
Explanation clone as described earlier circulation exchange according to manufacturer, the L27V variant CP84 of direct montage and CP103(N-(SS-169)-(1-SS1)-FRB-C and C-(1-SS1)-(SS-169)-FKBP) and at rabbit reticulocyte lysate (RRL；Promega Corp.) middle expression (25 μ L).By expression response and the 2X knot of 10 μ L of 1.25 μ L of each dimerization pair Close buffer (100mM HEPES, 200mM NaCl, 0.2%CHAPS, 2mM EDTA, 20% glycerol, 20mM DTT, pH7.5) Mix with 7.5 μ L aqueous phases, and 18 μ L are transferred in the hole of 96 hole flat boards.2 μ L rapamycin (final concentrations 0 are added to this reaction And 0.1-1000nM), and at room temperature incubation reaction 10 minutes.After hatching, (50X stock solution exists to add 100 μ L PBI-3939 Measure in buffer and be diluted to 1X) and at room temperature hatch 3 minutes.?Luminous (figure is measured on photometer 87B) and determine response (Figure 87 C).Figure 87 B-C proves that the OgLuc variant of the present invention can be used for by PCA-type dimerization mensuration Detection protein-protein interaction.
Embodiment 58 intracellular half life
Determine the intracellular half life of OgLuc variant 9B8,9B8+K33N, V2, L27V and V2+L27M.According to manufacturer Explanation utilize(Mirus) utilize containing 9B8,9B8+K33N, V2, L27V (" V2+L27V ") or V2+ In the 30 μ L100ng/ μ L plasmid DNA transfection 15-100mm flat boards of L27M (all in pF4A vector background) containing 10%FBS and Chinese hamster ovary celI (500,000) in the F12 culture medium of 1X Sodium Pyruvate.Then by cell incubation 6 hours.
After hatching, remove culture medium and add 1mL pancreatin to be departed from from flat board by cell.Then the F12 adding 3mL cultivates Base, and count cell.Then by cell with 10,000 cells/well be inoculated in 6 holes of 96 hole flat boards (6 holes/variant) and Overnight incubation at 37 DEG C.By 3 flat boards of sample allocation overlay.Each flat board has 6 repetitions of different point in time measurement.
After night incubation, from cell, remove culture medium be used for t=0 sample, and add 100 μ L mensuration buffer (previously Described；Without substrate).Freezing sample be stored in-20 DEG C on dry ice.?Middle by cycloheximide (100mg/mL) it is diluted to final concentration 1mg/mL at 1: 100.Exist equallyMiddle by DMSO (100%) 1: 100 dilution (final concentration 1%).The cycloheximide (1mg/mL) (11 μ L) of dilution is added to 3 repetitions of the variant sample of each transfection In and the DMSO (1%) of the dilution of 11 μ L added to other 3 repeat.Then at 37 DEG C, 5%CO2Lower incubated cell is also Remove and process at different time points (that is, 0,0.5,0.9,2.5,4.3 and 6.2 hour) as t=0 sample.
In order to analyze, cell be melted to room temperature, and measure 10 μ L in 50 μ L measure reagent.?Light Luminescence is measured on degree meter.At each time point, luminescence measured by the sample processed about untreated and cycloheximide.By not The RLU of the RLU of the cell the processed cell to utilizing cycloheximide to process is standardized.
At each time point by measuring the ratio of luminescence and the untreated luminescence processed from cycloheximide (CHX) Calculate the intracellular half life of each variant.Then draw and elapse the natural logrithm of ratio in time (ratio of process is untreated %), and calculate the half-life (table 46).OgLuc variant utilizes the cell that the CMV promoter of full strength has about 6-9 hour The interior half-life, but utilize CMV deletion mutants (d2) half-life have dropped.The PEST fall combined with the CMV promoter of full strength The existence solving signal significantly reduces the half-life.
Table 46
Sample CMV is without degraded signal CMV d2 is without degraded signal CMV Pest
9B8 6.32 3.87 1.43
K33N 9.24 3.70 1.18
V2 9.63 4.28 1.61
V2+L27V 6.66 4.78 1.63
V2+L27M 8.89 6.98 1.63
(data are not shown to utilize HEK293 cell to utilize the reverse transfection procedure described in embodiment 52 to complete another experiment Go out).Result from this experiment shows that the intracellular half life with the L27V variant of PEST is 10 minutes.Used by this experiment Without degraded signal L27V variant during this experiment, do not show decay.Decay in this case about during t=0 Untreated cell standard.
Embodiment 59 OgLuc variant is exposed to carbamide
Owing to known LUC Photinus pyralis LUC Photinus pyralis FL is relatively unstable, it exposes for carbamide is more sensitive.For really The most fixed be also this situation for OgLuc variant, it is determined that the OgLuc sensitivity to carbamide.By 45.3 μMs of L27V enzymes of 5 μ l Urea liquid (100mM MOPS, pH7.2,100mM NaCl, 1mM CDTA, 5% glycerol and the urine of variable concentrations with 100 μ L Element) mix mutually and at room temperature hatch 30 minutes.Carbamide+L27V the enzymatic solution 10,000 of 5 μ L is diluted to again without phenol red+ 0.1%DMEM in, by 50 μ L and containing 100 μMs of PBI-3939 50 μ L measure reagent (previously described ) mix mutually when being incorporated in 10 minutes and read luminous (Figure 88).Figure 88 shows L27V opposing carbamide or is removing after carbamide as quick as thought Refolding is functional enzyme.This shows when relating to chemical modification condition, such as, uses before reaction based on OgLuc variant MULTIPLE COMPOSITE in the condition that degeneration stops enzyme reaction, L27V can be used as reporting sub-enzyme.
The L27V variant stock solution 100,000 of the purification of 0.31mg/mL is diluted to buffer (PBS+1mM DTT+ again Hatch together with 3M carbamide 30 minutes in 0.005%IGEPAL) and at 25 DEG C, and then with containing 100 μMs of PBI-3939 (first Before described) mensuration reagent 1: 1 (50 μ L+50 μ L) mix mutually.Exist as described earlier Measure reaction on F500 photometer and (continue 100 minutes；1 minute reading time interval) (Figure 89).Result shows 3M Carbamide reduces the activity about 50% of L27V variant, but after 2 times of carbamide of dilution (to 1.5M final concentration), activity increases, may Owing to refolding is caused.
The imaging of embodiment 60 OgLuc fusion protein
This example indicates OgLuc and OgLuc variant and swashs without fluorescence for monitoring protein translocation in living cells The purposes sent out.OgLuc variant and human glucocorticoid receptor (GR；SEQ ID NO:451 and 452), human kinase protein C α (PKCa；SEQ ID NO:449 and 450) or LC3 (SEQ ID NO:577 and 578) blend.For utilizing biodiversity resources to divide Analysis sub-cellular protein indexing, by HeLa cell with 2x104Individual cell/cm2It is inoculated into containing in culture vessel with glass bottom (MatTek) In the DMEM culture medium of 10%FBS.Then at 37 DEG C, 24 hours under 5%CO2, are hatched.Then utilize 1/20 volume transfection (HD and be cloned in pF5A carrier (Promega Corp.) coding L27V02-GR (SEQ ID NO:453 and 454) or the DNA of L27V02-PKC α (SEQ ID NO:455 and 456)) mixture transfectional cell.Plasmid for L27V02-GR DNA1: 20 are diluted in pGEM-3ZF (Promega Corp.) obtain the expression of suitable L27V02-GR.About The DNA of L27V02-LC3 and L27V02-PKC α uses undiluted.Then at 37 DEG C, 5%CO2Lower incubated cell 24 hours.Profit The cell that GR fusion protein will be utilized to transfect by the MEM culture medium (Invitrogen) supplementing the FBS that 1% charcoal/glucosan processes To GR agonist hungry 20 hours.Transfect latter 24 hours (PKC α is measured) or transfect latter 48 hours (GR is measured), Immediately with being independent of CO containing 100 μMs of PBI-3939 before imaging2Culture medium substitute cell culture medium.At Olympus LV200 150X eyepiece is utilized to catch unfiltered image immediately on bioluminescence microscope.
The cytosol of L27V02-GR fusion protein is realized to core by stimulating 15 minutes with 0.5mM dexamethasone Indexing.Cytosol the turning to plasma membrane of L27V02-PKC alpha fusion protein is realized by utilizing 100nM PMA to stimulate 20 minutes Position.The cell of L27V02-LC3 fusion protein transfection does not carries out the DMEM culture medium processing or utilizing containing 10%FBS (Invitrogen) the 50mM chloroquine in processes.
L27V02-glucocorticoid receptor (GR)
When there is not glucocorticoid, glucocorticoid receptor (GR) (GR) and Hsp90 albumen are combined and are positioned at cytosol In.After GR interacts with glucocorticoid such as dexamethasone, GR albumen departs from from these albumen compositions and indexing is to core Express with regulator gene.Figure 90 A-B shows the L27V02-utilizing PBI-3939 substrate induced by dexamethasone in HeLa cell The cytosol of glucocorticoid receptor (GR) (GR) fusion protein is to the biodiversity resources of nuclear receptor (NR) indexing.
L27V02-PKCa
After processing with phorbol exters, PKC α albumen is raised plasma membrane and regulates cell response, including film kinetics and signal Transduction.Figure 91 A-B shows the phorbol exters utilizing PBI3939 substrate OgLuc L27V02-PKC α fusant in U-2OS cell Protein kinase C α (PCK α) cytosol of induction is to the biodiversity resources of plasma membrane translocation.
L27V-LC3
Finished LC-3 albumen and the significant step representing in autophagy of dissociating of autophagosome.Chloroquine processes and makes autophagy become Change and stagnate in this stage, result in LC-3 albumen on autophagosome, accumulate (producing point-like subcellular proteomics).Figure 92 A-B shows Two representational HeLa cell samples utilize PBI-3939 substrate OgLuc L27V-LC3 fusion protein (SEQ ID NO: 592 and 593) biodiversity resources of the autophagosome protein translocation of chloroquine induction.
Table 47 subordinate list
Sequence table
The thin Fructus Gleditsia acupuncture shrimp of SEQIDNO:1 (natural ripe OgLuc albumen)
FTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQMGLIEMIF KVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIYDERLINPDGSLLFRVT INGVTGWRLCENILA
SEQ ID NO:2 (C1A4E nucleotide)
ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTGTTAGAACA AGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATTGTGCTGTCTGGGGAGA ATGGGTTAAAATTTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGTTTTCAAATGGGTCTGATTGAAATG ATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGATTATTCTCCATTATGGTACACTCGTTATTGACGG TGTGACACCAAACATGATTGACTACTTTGGACGCCCTTACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAG TTACTGGAACTCTGTGGAACGGCAACAAGATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGC GTTACTATCAATGGAGTCACCGGATGGCGCCTTTGCGAGCGTATTCTTGCC
SEQ ID NO:3 (C1A4E albumen)
MVFTLEDFVGDWRQTAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKFDIHVIIPYEGLSGFQMGLIEM IFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:4 (QC27 nucleotide)
ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCTAGATCAAGTGTTAGAACA AGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATTGTGCTGTCTGGGGAGA ATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGTTTTCAAATGGGTCTGATTGAAATG ATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGATTATTCACCATTATGGTACACTCGTTATTGACGG TGTGACACCAAACATGATTGACTTCTTTGGACGCCCTTACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAG TTACTGGAACTCTGTGGAACGGCAACAAGATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGC GTTACTATCAATGGAGTCACCGGATGGCGCCTTTGCGAGCGTATTCTTGCC
SEQ ID NO:5 (QC27 albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEM IFKVVYPVDDHHFKIIHHYGTLVIDGVTPNMIDFFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:6 (QC279a nucleotide)
ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCTAGATCAACTGTTAGAACA AGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATTGTGCTGTCTGGGGAGA ATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGTTATCAAATGGGTCAGATTGAAAAG ATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGATTATTCGCCATTATGGTACACTCGTTATTGACGG TGTGACACCAAACATGATTGACTTCTTTGGACGCCCTTACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAG TTACTGGAACTCTGTGGAACGGCAACAAGATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGC GTTACTATCAATGGAATCACCGGATGGCGCCTTTGCGAGCGTATTCTTGCC
SEQ ID NO:7 (QC279a albumen)
MVFTLEDFVGDWRQTAGYNLDQLLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKIIRHYGTLVIDGVTPNMIDFFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGITGWRLCERILA
SEQ ID NO:8 (IVY nucleotide)
ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTGTTAGAACA AGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATTGTGCTGTCTGGGGAGA ATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGTTTTCAAATGGGTCTGATTGAAATG ATCTACAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTTATTCTCCATTATGGTACACTCGTTATTGACGG TGTGACACCAAACATGATTGACTACTTTGGACGCCCTTACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAG TTACTGGAACTCTGTGGAACGGCAACAAGATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGC GTTACTATCAATGGAGTCACCGGATGGCGCCTTTGCGAGCGTATTCTTGCC
SEQ ID NO:9 (IVY albumen)
MVFTLEDFVGDWRQTAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEM IYKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:10 (IVY+C1.3 nucleotide)
ATGGTGTTTACATTGGAGGATTACGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTGTTAGAACA AGGAGGATTGTCTAGTCTGTTCCAAAATCTGGGAGTGTCTGTCACCCCAATCCAGAAAATTGTGCTGTCTGGGGAGA ATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGTTTTCAAATGGGTCTGATTGAAATG ATCTACAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTTATTCTCCATTATGGTACACTCGTTATTGACGG TGTGACACCAAACATGATTGACTACTTTGGACGCCCTTACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAG TTACTGGAACTCTGTGGAACGGCGACAAGATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGC GTTACTACCAATGGAGTCACCGGATGGCGCCTTTGCGAGCGTATTCTTGCC
SEQ ID NO:11 (IVY+C1.3 albumen)
MVFTLEDYVGDWRQTAGYNQDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEM IYKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGDKIIDERLINPDGSLLFR VTTNGVTGWRLCERILA
SEQ ID NO:12 (IVY C5.19 nucleotide)
ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTGTTAGAACA AGGAGGAGTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAATCACCCCAATCCAGAAAATTGTGCTGTCTGGGGAGA ATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGTTTTCAAATGGGTCTGATTGAAATG ATCTACAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTTATTCTCCATTATGGTACACTCGTTATTGACGG TGTGACACCAAACATGATTGACTACTTTGGACGCCCTTACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAG TTACTGGAACTCTGTGGAACGGCAACAAGATCATTGATGAGCGCCTGATCAACCCAGATGGTTCAATCCTCTTCCGC GTTACTATCAATGGAGTCACCGGATGGCGCCTTTGCGAGCGTATTCTTGCC
SEQ ID NO:13 (IVY C5.19 albumen)
MVFTLEDFVGDWRQTAGYNQDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEM IYKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSILFR VTINGVTGWRLCERILA
SEQ ID NO:14 (IV nucleotide)
ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTGTTAGAACA AGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATTGTGCTGTCTGGGGAGA ATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGTTTTCAAATGGGTCTGATTGAAATG ATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTTATTCTCCATTATGGTACACTCGTTATTGACGG TGTGACACCAAACATGATTGACTACTTTGGACGCCCTTACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAG TTACTGGAACTCTGTGGAACGGCAACAAGATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGC GTTACTATCAATGGAGTCACCGGATGGCGCCTTTGCGAGCGTATTCTTGCC
SEQ ID NO:15 (IV albumen)
MVFTLEDFVGDWRQTAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEM IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:16 (15C1 nucleotide)
ATGGTGTTTACATTGAAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTGTTAGAACA AGGAGGATTGTCTAGTCTGTTCCAAAATCTGGGAGTGTCAGTCACCCCAATCCAGAAAATTGTGCTGTCTGGGGAGA ATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGTTATCAAATGGGTCAGATTGAAAAG ATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTTATTCTCCATTATGGTACACTCGTTATTGACGG TGTGACACCAAACATGATTGACTACTTTGGACGCCCTTACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAG TTACTGGAACTCTGTGGAACGGCAACAAGATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGC GTTACTATCAATGGAGTCACCGGATGGCGCCTTTGCGAGCGTATTCTTGCC
SEQ ID NO:17 (15C1 albumen)
MVFTLKDFVGDWRQTAGYNQDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:18 (9B8 nucleotide)
ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCTAGATCAAGTGTTAGAACA AGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATTGTGCTGTCTGGGGAGA ATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGTTATCAAATGGGTCAGATTGAAAAG ATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTTATTCTCCATTATGGTACACTCGTTATTGACGG TGTGACACCAAACATGATTGACTACTTTGGACGCCCTTACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAG TTACTGGAACTCTGTGGAACGGCAACAAGATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGC GTTACTATCAATGGAGTCACCGGATGGCGCCTTTGCGAGCGTATTCTTGCC
SEQ ID NO:19 (9B8 albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:20 (9F6 nucleotide)
ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCTAGATCAAGTGTTAGAACA AGGAGGAGTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAATCACCCCAATCCAGAAAATTGTGCTGTCTGGGGAGA ATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGTTATCAAATGGGTCAGATTGAAAAG ATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTTATTCTCCATTATGGTACACTCGTTATTGACGG TGTGACACCAAACATGATTGACTACTTTGGACGCCCTTACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAG TTACTGGAACTCTGTGGAACGGCAACAAGATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGC GTTACTATCAATGGAGTCACCGGATGGCGCCTTTGCGAGCGTATTCTTGCC
SEQ ID NO:21 (9F6 albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:22 (IV opt nucleotide)
atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaaccaagaccaagtccttgagca gggcggtctgtccagtttgtttcagaaactcggggtgtccgtaacaccgatccaaaagattgtcctgagcggtgaaa acggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcttccagatgggcctcattgagatg atctttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgc gtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg
SEQ ID NO:23 (IV optB nucleotide)
atggtattcacactggaggattttgtcggtgattggcggcaaaccgctgggtacaaccaggaccaggttctcgaaca agggggcctcagctccctgtttcaaaaactgggtgttagcgttacacctattcaaaaaatcgtgctctccggggaaa acgggctcaaaatcgatattcatgtgattatcccttacgaagggctctccgggtttcagatggggctgatcgaaatg atctttaaggtcgtctatcccgtagatgatcaccacttcaaggtgatcctccactacgggaccctcgtaattgatgg cgtgacccccaacatgatcgactattttgggcgcccttacgaggggattgctgtcttcgatggcaaaaaaattacag tgacaggcacactctggaacgggaataagatcattgatgagcgcctgattaatcccgatgggagcctgctctttcgg gtgacaattaacggcgtaacaggctggcgcctctgtgaacggattctggcg
SEQ ID NO:24 (9B8opt nucleotide)
atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagca gggcggtctgtccagtttgtttcagaaactcggggtgtccgtaacaccgatccaaaagattgtcctgagcggtgaaa acggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggctatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgc gtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg
SEQ ID NO:25 (9B8optB nucleotide)
atggtattcacactggaggattttgtcggtgattggcggcaaaccgctgggtacaacctcgaccaggttctcgaaca agggggcctcagctccctgtttcaaaaactgggtgttagcgttacacctattcaaaaaatcgtgctctccggggaaa acgggctcaaaatcgatattcatgtgattatcccttacgaagggctctccgggtatcagatggggcagatcgaaaaa atctttaaggtcgtctatcccgtagatgatcaccacttcaaggtgatcctccactacgggaccctcgtaattgatgg cgtgacccccaacatgatcgactattttgggcgcccttacgaggggattgctgtcttcgatggcaaaaaaattacag tgacaggcacactctggaacgggaataagatcattgatgagcgcctgattaatcccgatgggagcctgctctttcgg gtgacaattaacggcgtaacaggctggcgcctctgtgaacggattctggcg
SEQ ID NO:26 (8A3 nucleotide)
Atggtgattacattggaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtgttagaaca aggaggagtgtctagtctgttccaaaagctgggagtgtcaatcaccccaatccagaaaattgtgctgtctggggaga atgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggttttcaaatgggtctgattgaaatg atcttcaaagttgtttacccagtggatgatcatcatttcaaggttattctccattatggtacactcgttattgacgg tgtgacaccaaacatgattgactactttggacgcccttacgagggaattgctgtgtttgacggcaagaagatcacag ttactggaactctgtggaacggcaacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgc gttactatcaatggagtcaccggatggcgcctttgcgagcgtattcttgcc
SEQ ID NO:27 (8A3 albumen)
MVITLEDFVGDWRQTAGYNQDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEM IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:28 (Luc2 nucleotide)
atggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggcgagcagct gcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacatatcgaggtggacatta cctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgctatgggctgaatacaaaccatcgg atcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtgttgggtgccctgttcatcggtgtggctgtggc cccagctaacgacatctacaacgagcgcgagctgctgaacagcatgggcatcagccagcccaccgtcgtattcgtga gcaagaaagggctgcaaaagatcctcaacgtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagc aagaccgactaccagggcttccaaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacga cttcgtgcccgagagcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgccca agggcgtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccagatc atccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctgggctacttgat ctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagcttgcaagactataagattc aatctgccctgctggtgcccacactatttagcttcttcgctaagagcactctcatcgacaagtacgacctaagcaac ttgcacgagatcgccagcggcggggcgccgctcagcaaggaggtaggtgaggccgtggccaaacgcttccacctacc aggcatccgccagggctacggcctgacagaaacaaccagcgccattctgatcacccccgaaggggacgacaagcctg gcgcagtaggcaaggtggtgcccttcttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaac cagcgcggcgagctgtgcgtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctct catcgacaaggacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggacc ggctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaacacccc aacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgcagtcgtcgtgctgga acacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggttacaaccgccaagaagctgcgcg gtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaagttggacgcccgcaagatccgcgagattctc attaaggccaagaagggcggcaagatcgccgtt
SEQ ID NO:29 (Luc2 albumen)
medaknikkgpapfypledgtageqlhkamkryalvpgtiaftdahievdityaeyfemsvrlaeamkryglntnhr ivvcsenslqffmpvlgalfigvavapandiynerellnsmgisqptvvfvskkglqkilnvqkklpiiqkiiimds ktdyqgfqsmytfvtshlppgfneydfvpesfdrdktialimnssgstglpkgvalphrtacvrfshardpifgnqi ipdtailsvvpfhhgfgmfttlgylicgfrvvlmyrfeeelflrslqdykiqsallvptlfsffakstlidkydlsn lheiasggaplskevgeavakrfhlpgirqgygltettsailitpegddkpgavgkvvpffeakvvdldtgktlgvn qrgelcvrgpmimsgyvnnpeatnalidkdgwlhsgdiaywdedehffivdrlkslikykgyqvapaelesillqhp nifdagvaglpdddagelpaavvvlehgktmtekeivdyvasqvttakklrggvvfvdevpkgltgkldarkireil ikakkggkiav
SEQ ID NO:30 (HRL (humanized sea pansy genus) nucleotide)
Atggcttccaaggtgtacgaccccgagcaacgcaaacgcatgatcactgggcctcagtggtgggctcgctgcaagca aatgaacgtgctggactccttcatcaactactatgattccgagaagcacgccgagaacgccgtgatttttctgcatg gtaacgctgcctccagctacctgtggaggcacgtcgtgcctcacatcgagcccgtggctagatgcatcatccctgat ctgatcggaatgggtaagtccggcaagagcgggaatggctcatatcgcctcctggatcactacaagtacctcaccgc ttggttcgagctgctgaaccttccaaagaaaatcatctttgtgggccacgactggggggcttgtctggcctttcact actcctacgagcaccaagacaagatcaaggccatcgtccatgctgagagtgtcgtggacgtgatcgagtcctgggac gagtggcctgacatcgaggaggatatcgccctgatcaagagcgaagagggcgagaaaatggtgcttgagaataactt cttcgtcgagaccatgctcccaagcaagatcatgcggaaactggagcctgaggagttcgctgcctacctggagccat tcaaggagaagggcgaggttagacggcctaccctctcctggcctcgcgagatccctctcgttaagggaggcaagccc gacgtcgtccagattgtccgcaactacaacgcctaccttcgggccagcgacgatctgcctaagatgttcatcgagtc cgaccctgggttcttttccaacgctattgtcgagggagctaagaagttccctaacaccgagttcgtgaaggtgaagg gcctccacttcagccaggaggacgctccagatgaaatgggtaagtacatcaagagcttcgtggagcgcgtgctgaag aacgagcag
SEQ ID NO:31 (HRL (humanized sea pansy genus) albumen)
MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPD LIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWD EWPDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKP DVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLK NEQ
SEQ ID NO:32 (Id-HRL-HT7 sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggcttccaaggtgtacgaccccgagcaacgcaaacgcatg atcactgggcctcagtggtgggctcgctgcaagcaaatgaacgtgctggactccttcatcaactactatgattccga gaagcacgccgagaacgccgtgatttttctgcatggtaacgctgcctccagctacctgtggaggcacgtcgtgcctc acatcgagcccgtggctagatgcatcatccctgatctgatcggaatgggtaagtccggcaagagcgggaatggctca tatcgcctcctggatcactacaagtacctcaccgcttggttcgagctgctgaaccttccaaagaaaatcatctttgt gggccacgactggggggcttgtctggcctttcactactcctacgagcaccaagacaagatcaaggccatcgtccatg ctgagagtgtcgtggacgtgatcgagtcctgggacgagtggcctgacatcgaggaggatatcgccctgatcaagagc gaagagggcgagaaaatggtgcttgagaataacttcttcgtcgagaccatgctcccaagcaagatcatgcggaaact ggagcctgaggagttcgctgcctacctggagccattcaaggagaagggcgaggttagacggcctaccctctcctggc ctcgcgagatccctctcgttaagggaggcaagcccgacgtcgtccagattgtccgcaactacaacgcctaccttcgg gccagcgacgatctgcctaagatgttcatcgagtccgaccctgggttcttttccaacgctattgtcgagggagctaa gaagttccctaacaccgagttcgtgaaggtgaagggcctccacttcagccaggaggacgctccagatgaaatgggta agtacatcaagagcttcgtggagcgcgtgctgaagaacgagcaggtttctctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcctgggcga gcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacctcctcct acgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtatgggcaaa tccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatggatgccttcatcgaagccctgggtct ggaagaggtcgtcctggtcattcacgactggggctccgctctgggtttccactgggccaagcgcaatccagagcgcg tcaaaggtattgcatttatggagttcatccgccctatcccgacctgggacgaatggccagaatttgcccgcgagacc ttccaggccttccgcaccaccgacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgcc gatgggtgtcgtccgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcg agccactgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaatac atggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccggccga agccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctgcaagaag acaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatttccggt
SEQ ID NO:33 (Id-HRL-HT7 sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMASKVYDPEQRKRM ITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVARCIIPDLIGMGKSGKSGNGS YRLLDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAESVVDVIESWDEWPDIEEDIALIKS EEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLR ASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQVSLEPTTEDLY FQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGK SDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARET FQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEY MDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG
SEQ ID NO:34 (8A3-HT7 fusion nucleus thuja acid)
atggtgattacattggaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtgttagaaca aggaggagtgtctagtctgttccaaaagctgggagtgtcaatcaccccaatccagaaaattgtgctgtctggggaga atgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggttttcaaatgggtctgattgaaatg atcttcaaagttgtttacccagtggatgatcatcatttcaaggttattctccattatggtacactcgttattgacgg tgtgacaccaaacatgattgactactttggacgcccttacgagggaattgctgtgtttgacggcaagaagatcacag ttactggaactctgtggaacggcaacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgc gttactatcaatggagtcaccggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgagga tctgtactttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcc tgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacc tcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtat gggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatggatgccttcatcgaagccc tgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctgggtttccactgggccaagcgcaatcca gagcgcgtcaaaggtattgcatttatggagttcatccgccctatcccgacctgggacgaatggccagaatttgcccg cgagaccttccaggccttccgcaccaccgacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggta cgctgccgatgggtgtcgtccgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgtt gaccgcgagccactgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcga agaatacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccac cggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctg caagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatttccggt
SEQ ID NO:35 (8A3-HT7 fusion protein)
MVITLEDFVGDWRQTAGYNQDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEM IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPT SSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNP ERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPV DREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLL QEDNPDLIGSEIARWLSTLEISG
SEQ ID NO:36 (9B8-HT7 fusion nucleus thuja acid)
atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaacctagatcaagtgttagaaca aggaggattgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccagaaaattgtgctgtctggggaga atgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggttatcaaatgggtcagattgaaaag atcttcaaagttgtttacccagtggatgatcatcatttcaaggttattctccattatggtacactcgttattgacgg tgtgacaccaaacatgattgactactttggacgcccttacgagggaattgctgtgtttgacggcaagaagatcacag ttactggaactctgtggaacggcaacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgc gttactatcaatggagtcaccggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgagga tctgtactttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcc tgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacc tcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtat gggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatggatgccttcatcgaagccc tgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctgggtttccactgggccaagcgcaatcca gagcgcgtcaaaggtattgcatttatggagttcatccgccctatcccgacctgggacgaatggccagaatttgcccg cgagaccttccaggccttccgcaccaccgacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggta cgctgccgatgggtgtcgtccgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgtt gaccgcgagccactgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcga agaatacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccac cggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctg caagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatttccggt
SEQ ID NO:37 (9B8-HT7 fusion protein)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPT SSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNP ERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPV DREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLL QEDNPDLIGSEIARWLSTLEISG
SEQ ID NO:38 (9F6-HT7 fusion nucleus thuja acid)
atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaacctagatcaagtgttagaaca aggaggagtgtctagtctgttccaaaagctgggagtgtcaatcaccccaatccagaaaattgtgctgtctggggaga atgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggttatcaaatgggtcagattgaaaag atcttcaaagttgtttacccagtggatgatcatcatttcaaggttattctccattatggtacactcgttattgacgg tgtgacaccaaacatgattgactactttggacgcccttacgagggaattgctgtgtttgacggcaagaagatcacag ttactggaactctgtggaacggcaacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgc gttactatcaatggagtcaccggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgagga tctgtactttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcc tgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacc tcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtat gggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatggatgccttcatcgaagccc tgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctgggtttccactgggccaagcgcaatcca gagcgcgtcaaaggtattgcatttatggagttcatccgccctatcccgacctgggacgaatggccagaatttgcccg cgagaccttccaggccttccgcaccaccgacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggta cgctgccgatgggtgtcgtccgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgtt gaccgcgagccactgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcga agaatacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccac cggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctg caagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatttccggt
SEQ ID NO:39 (9F6-HT7 fusion protein)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPT SSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNP ERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPV DREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLL QEDNPDLIGSEIARWLSTLEISG
SEQ ID NO:40 (Id-9B8opt-HT7 sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtctgtccagtttgtttcagaaactcggggtgtccgt aacaccgatccaaaagattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaag gtctgagcggctatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatga aggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagc gcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgt attcttgccggatatctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgc tggctgtctactctggagatttccggtt
SEQ ID NO:41 (Id-9B8opt-HT7 sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWRQ TAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIAR WLSTLEISG
SEQ ID NO:42 (9B8opt+K33N nucleotide)
Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagca gggcggtctgtccagtttgtttcagaatctcggggtgtccgtaacaccgatccaaaagattgtcctgagcggtgaaa acggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggctatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgc gtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg
SEQ ID NO:43 (9B8opt+K33N albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:44 (QC27-HT7 nucleotide)
atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaacctagatcaagtgttagaaca aggaggattgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccagaaaattgtgctgtctggggaga atgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggttttcaaatgggtctgattgaaatg atcttcaaagttgtttacccagtggatgatcatcatttcaagattattcaccattatggtacactcgttattgacgg tgtgacaccaaacatgattgacttctttggacgcccttacgagggaattgctgtgtttgacggcaagaagatcacag ttactggaactctgtggaacggcaacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgc gttactatcaatggagtcaccggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgagga tctgtactttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcc tgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacc tcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtat gggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatggatgccttcatcgaagccc tgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctgggtttccactgggccaagcgcaatcca gagcgcgtcaaaggtattgcatttatggagttcatccgccctatcccgacctgggacgaatggccagaatttgcccg cgagaccttccaggccttccgcaccaccgacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggta cgctgccgatgggtgtcgtccgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgtt gaccgcgagccactgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcga agaatacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccac cggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctg caagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatttccggt
SEQ ID NO:45 (QC27-HT7 albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEM IFKVVYPVDDHHFKIIHHYGTLVIDGVTPNMIDFFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPT SSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNP ERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPV DREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLL QEDNPDLIGSEIARWLSTLEISG
SEQ ID NO:46(8F2)
atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtgttagaaca aggaggagtgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccagaaaattgtgctgtctggggaga atgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggttttcaaatgggtctgattgaaatg atcttcaaagttgtttacccagtggatgatcatcatttcaaggttattctccattatggtacactcgttattgacgg tgtgacaccaaacatgattgactactttggacgcccttacgagggaattgctgtgtttgacggcaagaagatcacag ttactggaactctgtggaacggcaacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgc gttactatcaatggagtcaccggatggcgcctttgcgagcgtattcttgcc
SEQ ID NO:47 (8F2 albumen)
MVFTLEDFVGDWRQTAGYNQDQVLEQGGVSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEM IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:48 (IV-HT7 nucleotide)
atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtgttagaaca aggaggattgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccagaaaattgtgctgtctggggaga atgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggttttcaaatgggtctgattgaaatg atcttcaaagttgtttacccagtggatgatcatcatttcaaggttattctccattatggtacactcgttattgacgg tgtgacaccaaacatgattgactactttggacgcccttacgagggaattgctgtgtttgacggcaagaagatcacag ttactggaactctgtggaacggcaacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgc gttactatcaatggagtcaccggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgagga tctgtactttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcc tgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacc tcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtat gggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatggatgccttcatcgaagccc tgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctgggtttccactgggccaagcgcaatcca gagcgcgtcaaaggtattgcatttatggagttcatccgccctatcccgacctgggacgaatggccagaatttgcccg cgagaccttccaggccttccgcaccaccgacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggta cgctgccgatgggtgtcgtccgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgtt gaccgcgagccactgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcga agaatacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccac cggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctg caagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatttccggt
SEQ ID NO:49 (IV-HT7 albumen)
MVFTLEDFVGDWRQTAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEM IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPT SSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNP ERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPV DREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLL QEDNPDLIGSEIARWLSTLEISG
SEQ ID NO:50 (8F2-HT7 nucleotide)
atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtgttagaaca aggaggagtgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccagaaaattgtgctgtctggggaga atgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggttttcaaatgggtctgattgaaatg atcttcaaagttgtttacccagtggatgatcatcatttcaaggttattctccattatggtacactcgttattgacgg tgtgacaccaaacatgattgactactttggacgcccttacgagggaattgctgtgtttgacggcaagaagatcacag ttactggaactctgtggaacggcaacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgc gttactatcaatggagtcaccggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgagga tctgtactttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcc tgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacc tcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtat gggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatggatgccttcatcgaagccc tgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctgggtttccactgggccaagcgcaatcca gagcgcgtcaaaggtattgcatttatggagttcatccgccctatcccgacctgggacgaatggccagaatttgcccg cgagaccttccaggccttccgcaccaccgacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggta cgctgccgatgggtgtcgtccgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgtt gaccgcgagccactgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcga agaatacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccac cggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctg caagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatttccggt
SEQ ID NO:51 (8F2-HT7 albumen)
MVFTLEDFVGDWRQTAGYNQDQVLEQGGVSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEM IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPT SSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNP ERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPV DREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLL QEDNPDLIGSEIARWLSTLEISG
SEQ ID NO:52 (15C1-HT7 nucleotide)
atggtgtttacattgaaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtgttagaaca aggaggattgtctagtctgttccaaaatctgggagtgtcagtcaccccaatccagaaaattgtgctgtctggggaga atgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggttatcaaatgggtcagattgaaaag atcttcaaagttgtttacccagtggatgatcatcatttcaaggttattctccattatggtacactcgttattgacgg tgtgacaccaaacatgattgactactttggacgcccttacgagggaattgctgtgtttgacggcaagaagatcacag ttactggaactctgtggaacggcaacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgc gttactatcaatggagtcaccggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgagga tctgtactttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcc tgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacc tcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtat gggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatggatgccttcatcgaagccc tgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctgggtttccactgggccaagcgcaatcca gagcgcgtcaaaggtattgcatttatggagttcatccgccctatcccgacctgggacgaatggccagaatttgcccg cgagaccttccaggccttccgcaccaccgacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggta cgctgccgatgggtgtcgtccgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgtt gaccgcgagccactgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcga agaatacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccac cggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctg caagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatttccggt
SEQ ID NO:53 (15C1-HT7 albumen)
MVFTLKDFVGDWRQTAGYNQDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPT SSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNP ERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPV DREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLL QEDNPDLIGSEIARWLSTLEISG
The thin Fructus Gleditsia acupuncture shrimp of SEQ ID NO:54 (OgLuc secreting signal peptide)
atggcttactccacactgttcatcattgctctcacagccgtcgtaacacaagcctcctccacacagaaaagcaacct gaca
SEQ ID NO:55 (Id-C1A4E-HT7 sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacattggaggatttcgttggagactggcggcag acagctggatacaaccaagatcaagtgttagaacaaggaggattgtctagtctgttccaaaagctgggagtgtcagt caccccaatccagaaaattgtgctgtctggggagaatgggttaaaatttgatattcatgtcatcatcccttacgagg gactcagtggttttcaaatgggtctgattgaaatgatcttcaaagttgtttacccagtggatgatcatcatttcaag attattctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgcccttacga gggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaagatcattgatgagc gcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcaccggatggcgcctttgcgagcgt attcttgccggatctctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgc tggctgtctactctggagatttccggt
SEQ ID NO:56 (Id-C1A4E-HT7 sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWRQ TAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKFDIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFK IILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAGSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIAR WLSTLEISG
SEQ ID NO:57 (Id-IV-HT7 sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacattggaggatttcgttggagactggcggcag acagctggatacaaccaagatcaagtgttagaacaaggaggattgtctagtctgttccaaaagctgggagtgtcagt caccccaatccagaaaattgtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagg gactcagtggttttcaaatgggtctgattgaaatgatcttcaaagttgtttacccagtggatgatcatcatttcaag gttattctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgcccttacga gggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaagatcattgatgagc gcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcaccggatggcgcctttgcgagcgt attcttgccgtttctctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgc tggctgtctactctggagatttccggt
SEQ ID NO:58 (Id-IV-HT7 sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWRQ TAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGFQMGLIEMIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAVSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIAR WLSTLEISG
SEQ ID NO:59 (Id-9B8opt+K33N-HT7 sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgt aacaccgatccaaaagattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaag gtctgagcggctatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatga aggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagc gcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggatatctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgc tggctgtctactctggagatttccggt
SEQ ID NO:60 (Id-9B8opt+K33N-HT7 sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWRQ TAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIAR WLSTLEISG
SEQ ID NO:61 (Id-9B8-HT7 sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacattggaggatttcgttggagactggcggcag acagctggatacaacctagatcaagtgttagaacaaggaggattgtctagtctgttccaaaagctgggagtgtcagt caccccaatccagaaaattgtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagg gactcagtggttatcaaatgggtcagattgaaaagatcttcaaagttgtttacccagtggatgatcatcatttcaag gttattctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgcccttacga gggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaagatcattgatgagc gcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcaccggatggcgcctttgcgagcgt attcttgccgtttctctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgc tggctgtctactctggagatttccggt
SEQ ID NO:62 (Id-9B8-HT7 sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWRQ TAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAVSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIAR WLSTLEISG
SEQ ID NO:63 (Id-9F6-HT7 sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacattggaggatttcgttggagactggcggcag acagctggatacaacctagatcaagtgttagaacaaggaggagtgtctagtctgttccaaaagctgggagtgtcaat caccccaatccagaaaattgtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagg gactcagtggttatcaaatgggtcagattgaaaagatcttcaaagttgtttacccagtggatgatcatcatttcaag gttattctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgcccttacga gggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaagatcattgatgagc gcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcaccggatggcgcctttgcgagcgt attcttgccgtttctctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgc tggctgtctactctggagatttccggt
SEQ ID NO:64 (Id-9F6-HT7 sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWRQ TAGYNLDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAVSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIAR WLSTLEISG
SEQ ID NO:65 (9B8opt-P nucleotide)
atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagca gggcggtctgtccagtttgtttcagaaactcggggtgtccgtaacaccgatccaaaagattgtcctgagcggtgaaa acggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggctatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgc gtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgaattctcacggcttccctcccgaggt ggaggagcaggccgccggcaccctgcccatgagctgcgcccaggagagcggcatggatagacaccctgctgcttgcg ccagcgccaggatcaacgtc
SEQ ID NO:66(2X ARE)
TAGCTTGGAAATGACATTGCTAATGGTGACAAAGCAACTTTTAGCTTGGAAATGACATTGCTAATGGTGACAAAGCA ACTTT
SEQ ID NO:67(HRE)
CTGGAATTTTCTAGACTGGAATTTTCTAGACTGGAATTTTCTAGA
SEQ ID NO:68 (K33N+170G nucleotide)
atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagca gggcggtctgtccagtttgtttcagaatctcggggtgtccgtaacaccgatccaaaagattgtcctgagcggtgaaa acggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggctatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgc gtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggga
SEQ ID NO:69 (K33N+170G albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAG
SEQ ID NO:70 (27A5 (NF) nucleotide)
ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAGCA GGGCGGTCTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAAGATTGTCCTGAGCGGTGAAA ACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCTATCAGATGGGCCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATTCTGCACTATGGCACACTGGTAATCGACGG GGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTG TAACCGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGC GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCGGGA
SEQ ID NO:71 (27A5 (NF) albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAG
SEQ ID NO:72 (23D4 (NF) nucleotide)
ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAGCA GGGCGGCCTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACACCGATCCAAAAGATTGTCCTGAGCGGTGAAA ACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCTATCAGATGGGCCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATTCTGCACTATGGCACACTGGTAATCGACGG GGTTACGCCGAACTTGATCGACTATTTCGGACGTCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTG TAACCGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGC GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCGGGA
SEQ ID NO:73 (23D4 (NF) albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNLIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAG
SEQ ID NO:74 (24C2 (NF) nucleotide)
ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCAGCAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAGCA GGGCGGTCTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAAGATTGTCCTGAGCGGTGAAA ACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCTATCAGATGGGCCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATTCTGCACTATGGCACACTGGTAATCGACGG GGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTG TAACCGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGC GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCGGGA
SEQ ID NO:75 (24C2 (NF) albumen)
MVFTLEDFVGDWQQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAG
SEQ ID NO:76 (Id-23D4-HT7 sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggcctgtccagtttgtttcagaatctcggggtgtccgt aacaccgatccaaaagattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaag gtctgagcggctatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacttgatcgactatttcggacgtccgtatga aggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagc gcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggatatctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctg
SEQ ID NO:77 (Id-23D4-HT7 sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWRQ TAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNLIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGL
SEQ ID NO:78 (Id-24C2-HT7 sandwich merges glycosides thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacactcgaagatttcgtaggggactggcagcag acagccggctacaacctggaccaagtccttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgt aactccgatccaaaagattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaag gtctgagcggctatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatga aggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagc gcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggatatctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgc tggctgtctactctggagatttccggt
SEQ ID NO:79 (Id-24C2-HT7 sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWQQ TAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSGYQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIAR WLSTLEISG
SEQ ID NO:80 (1F7 (NF) nucleotide)
ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAGCA GGGCGGTCTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACACCGATCCAAAGGATTGTCCTGAGCGGTGAAA ACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGATCAGATGGGCCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATTCTGCACTATGGCACACTGGTAATCGACGG GGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTG TAACCGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGC GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCGGGA
SEQ ID NO:81 (1F7 (NF) albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAG
SEQ ID NO:82 (15H1 (NF) nucleotide)
ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGATCAAGTCCTTGAGCA GGGCGGTCTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACACCGATCCAAAAGATTGTCCTGAGCGGTGAAA ACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAACGGCTATCAGATGGGCCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATTCTGCACTATGGCACACTGGTAATCGACGG GGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTG TAACCGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGC GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCGGGA
SEQ ID NO:83 (15H1 (NF) albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLNGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAG
SEQ ID NO:84 (Id-1F7-HT7 sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgt aacaccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaag gtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatga aggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagc gcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggatatctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgc tggctgtctactctggag
SEQ ID NO:85 (Id-1F7-HT7 sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWRQ TAGYNLDQVLEQGGLSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIAR WLSTLEISG
SEQ ID NO:86 (Id-15H1-HT7 sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggatcaagtccttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgt aacaccgatccaaaagattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaag gtctgaacggctatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatga aggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagc gcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggatatctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgc tggctgtctactctggagatttccggt
SEQ ID NO:87 (Id-15H1-HT7 sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWRQ TAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLNGYQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIAR WLSTLEISG
SEQ ID NO:88 (9B8opt+K33N+L27V+T39T+K43R+Y68D nucleotide)
Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagca gggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa acggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgc gtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg
SEQ ID NO:89 (9B8opt+K33N+L27V+T39T+K43R+Y68D albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:90 (9B8opt+K33N+L27V+T39T+K43R+Y68D sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgt aactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaag gtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatga aggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagc gcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggatatctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgc tggctgtctactctggagatttccggt
SEQ ID NO:91 (9B8opt+K33N+L27V+T39T+K43R+Y68D sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWRQ TAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIAR WLSTLEISG
SEQ ID NO:92 (9B8opt+K33N+T39T+K43R+Y68D nucleotide)
Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagca gggcggtctgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa acggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgc gtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg
SEQ ID NO:93 (9B8opt+K33N+T39T+K43R+Y68D albumen)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:94 (9B8opt+K33N+T39T+K43R+Y68D sandwich fusion nucleus thuja acid)
atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctggacgagca gcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtgcccaccctgccccaga accgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagggacctgcagctggagctgaactcg gagtctgaagtcgggaccaccggaggccggggactgcctgtccgcgccccgctcagcaccctgaacggcgagatcag tgccttggcggccgaggcggcatgtgttccagccgacgatcgcatcttgtgtcgcgtttctcttgagaatctttatt ttcaggcgtctggaggtggtggcggagcgatcgccatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgt aactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaag gtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatga aggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagc gcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggatatctcgagccaaccactgaggatctgtactttcagagcgataacgatggatccgaaatcggtac tggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatg gcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccg acccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacga ccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggct ccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaa gctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtcgaga tggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaacgagctgccaatc gccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccagtcccctgtcccgaagct gctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgca aggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgc tggctgtctactctggagatttccggt
SEQ ID NO:95 (9B8opt+K33N+T39T+K43R+Y68D sandwich fusion protein)
MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIRDLQLELNS ESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGAIAMVFTLEDFVGDWRQ TAGYNLDQVLEQGGLSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAP THRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRP IPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPI AGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIAR WLSTLEISG
SEQ ID NO:96(CRE)
GCACCAGACAGTGACGTCAGCTGCCAGATCCCATGGCCGTCATACTGTGACGTCTTTCAGACACCCCTTGACGTCAA TGGGAGAACA
SEQ ID NO:97 (nucleotide CP84 non junction) Atggatgatcatcactttaaggtgattctgcactatggc acactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcga cggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacg gctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgatggtgttt acactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgt gtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctga agatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaag gtggtgtaccctgtg
SEQ ID NO:98 (PROTEIN C P84 non junction)
MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT GWRLCERILAMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL SGDQMGQIEKIFKVVYPV
SEQ ID NO:99 (nucleotide CP845AA joint)
Atggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcga ctatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacg gcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcggggagctccggtggaatggtgtttacactcgaagatttcgtagggga ctggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcg gggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg
SEQ ID NO:100 (PROTEIN C P845AA joint)
MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT GWRLCERILAGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVII PYEGLSGDQMGQIEKIFKVVYPV
SEQ ID NO:101 (nucleotide CP8410AA joint)
Atggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcga ctatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacg gcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcgggaagttctggtggagggagctccggtggaatggtgtttacactcga agatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtt tgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgac atccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgta ccctgtg
SEQ ID NO:102 (PROTEIN C P8410AA joint)
MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT GWRLCERILAGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKID IHVIIPYEGLSGDQMGQIEKIFKVVYPV
SEQ ID NO:103 (nucleotide CP8420AA joint)
Atggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcga ctatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacg gcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcgggaagttctggtggagggagctccggtggaggaagttctggtggagg gagctccggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggacc aagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtc ctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatggg ccagatcgaaaaaatttttaaggtggtgtaccctgtg
SEQ ID NO:104 (PROTEIN C P8420AA joint)
MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT GWRLCERILAGSSGGGSSGGGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIV LSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV
SEQ ID NO:105 (nucleotide CP95 non junction)
Atgggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgt gttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacc ccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgatg gtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcaggg cggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacg gcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatt tttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactat
SEQ ID NO:106 (PROTEIN C P95 non junction)
MGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAM VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKI FKVVYPVDDHHFKVILHY
SEQ ID NO:107 (nucleotide CP955AA joint)
Atgggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgt gttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacc ccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggg agctccggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggacca agtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcc tgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggc cagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactat
SEQ ID NO:108 (PROTEIN C P955AA joint)
MGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAG SSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMG QIEKIFKVVYPVDDHHFKVILHY
SEQ ID NO:109 (nucleotide CP9510AA joint)
Atgggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgt gttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacc ccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggga agttctggtggagggagctccggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccgg ctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccga tccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagc ggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattct gcactat
SEQ ID NO:110 (PROTEIN C P9510AA joint)
MGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAG SSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLS GDQMGQIEKIFKVVYPVDDHHFKVILHY
SEQ ID NO:111 (nucleotide CP9520AA joint)
Atgggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgt gttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacc ccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggga agttctggtggagggagctccggtggaggaagttctggtggagggagctccggtggaatggtgtttacactcgaaga tttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgt ttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatc catgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccc tgtggatgatcatcactttaaggtgattctgcactat
SEQ ID NO:112 (PROTEIN C P9520AA joint)
MGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAG SSGGGSSGGGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDI HVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHY
SEQ ID NO:113 (joint)
(GSGG)N
SEQ ID NO:114(L27V CP5TEV)
Atggatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccag tttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcg acatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtg taccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacat gatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgt ggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactac tgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaa
SEQ ID NO:115(L27V CP5TEV)
MDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVV YPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTING VTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLE
SEQ ID NO:116(L27V CP6TEV)
atgttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagttt gtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgaca tccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtac cctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgat cgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtgga acggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtg accggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactga gaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagat
SEQ ID NO:117(L27V CP6TEV)
MFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVY PVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV TGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLED
SEQ ID NO:118(L27V CP7TEV)
atggtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtt tcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatcc atgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcga ctatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacg gcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaa cttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttc
SEQ ID NO:119(L27V CP7TEV)
MVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYP VDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT GWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDF
SEQ ID NO:120(L27V CP9TEV)
atggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaa tctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtca tcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggat gatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactattt cggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaaca aaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctgg cggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgta cttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggg
SEQ ID NO:121(L27V CP9TEV)
MDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVD DHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVG
SEQ ID NO:122(L27V CP11TEV)
Atgcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcgg ggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcc cgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcat cactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacg gccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaatta tcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctg tgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttcca gagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactgg
SEQ ID NO:123(L27V CP11TEV)
MRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDH HFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRL CERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDW
SEQ ID NO:124(L27V CP12TEV)
Cagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtc cgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatg aaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgta tgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacg agcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgag cgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcga taacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggg
SEQ ID NO:125(L27V CP12TEV)
QTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHF KVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCE RILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVG
SEQ ID NO:126(L27V CP15TEV)
atgggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaac tccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtc tgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaagg catcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcc tgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcatt ttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacgg aagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagcc
SEQ ID NO:127(L27V CP15TEV)
MGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKV ILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERI LAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTA
SEQ ID NO:128(L27V CP18TEV)
ctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaag gattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatc agatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactat ggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgtt cgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccg acggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagt tctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtgg aggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaac
SEQ ID NO:129(L27V CP18TEV)
LDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHY GTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGS SGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYN
SEQ ID NO:130(L27V CP21TEV)
gtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcct gagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggcc agatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactg gtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaa aaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccc tgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtgga ggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttc tggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaa
SEQ ID NO:131(L27V CP21TEV)
VLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTL VIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGG GSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQ
SEQ ID NO:132(L27V CP24TEV)
cagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtga aaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaa aaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgac ggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcac tgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttcc gcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttct ggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaat ggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgag
SEQ ID NO:133(L27V CP24TEV)
QGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVID GVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSS GGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLE
SEQ ID NO:134(L27V CP27TEV)
gtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcct gaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaattttta aggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacg ccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgg gaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaacca tcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagag cctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttac actcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggt
SEQ ID NO:135(L27V CP27TEV)
VSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGE PTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGG
SEQ ID NO:136(L27V CP34TEV)
atgctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgt catcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtgg atgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactat ttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaa caaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggct ggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttg tacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtagg ggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaat
SEQ ID NO:137(L27V CP34TEV)
MLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDY FGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENL YFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQN
SEQ ID NO:138(L27V CP40TEV)
Atgccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaagg tctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaagg tgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaa ggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcg cctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgca ttttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataac ggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagc cggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaact
SEQ ID NO:139(L27V CP40TEV)
MPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYE GIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDN GSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVT
SEQ ID NO:140(L27V CP43TEV)
Atgaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcgg cgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgc actatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgcc gtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaa ccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgg gaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttct ggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaa cctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaa
SEQ ID NO:141(L27V CP43TEV)
MRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIA VFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSS GGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQ
SEQ ID NO:142(L27V CP44TEV)
Atgattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcga tcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcact atggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtg ttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccc cgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaa gttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggt ggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacct ggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaagg
SEQ ID NO:143(L27V CP44TEV)
MIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAV FDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSG GGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
SEQ ID NO:144(L27V CP45TEV)
Atggtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatca gatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatg gcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttc gacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccga cggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagtt ctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtgga ggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctgga ccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt
SEQ ID NO:145(L27V CP45TEV)
MVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVF DGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGG GSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
SEQ ID NO:146(L27V CP46TEV)
Atgctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagat gggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggca cactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgac ggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacgg ctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctg gtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggagga agttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggacca agtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt
SEQ ID NO:147(L27V CP46TEV)
MLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFD GKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGG SSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
SEQ ID NO:148(L27V CP47TEV)
atgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatggg ccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacac tggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc aaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctc cctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtg gaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagt tctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagt ccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctg
SEQ ID NO:149(L27V CP47TEV)
MSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDG KKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGS SGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVL
SEQ ID NO:150(L27V CP48TEV)
atgggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggcca gatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactgg taatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa aagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccct gctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggag gaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttct ggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtcct tgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcg
SEQ ID NO:151(L27V CP48TEV)
MGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGK KITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSS GGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLS
SEQ ID NO:152(L27V CP49TEV)
atggaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagat cgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaa tcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag atcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgct gttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaa gttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggt ggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttga gcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggt
SEQ ID NO:153(L27V CP49TEV)
MENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKK ITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSG GMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSG
SEQ ID NO:154(L27V CP50TEV)
atgaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcga aaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcg acggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatc actgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgtt ccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagtt ctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtgga atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagca gggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaa
SEQ ID NO:155(L27V CP50TEV)
MNGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKI TVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGG MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGE
SEQ ID NO:156(L27V CP51TEV)
atgggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaa aatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacg gggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcact gtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccg cgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctg gtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatg gtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcaggg cggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaac
SEQ ID NO:157(L27V CP51TEV)
MGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKIT VTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGM VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGEN
SEQ ID NO:158(L27V CP52TEV)
atgctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaat ttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacgggg ttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgta accgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgt aaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtg gagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtg tttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcgg tgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggc
SEQ ID NO:159(L27V CP52TEV)
MLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITV TGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMV FTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENG
SEQ ID NO:160(L27V CP53TEV)
atgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttt taaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggtta cgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacc gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaac catcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggag agcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgttt acactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgt gtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctg
SEQ ID NO:161(L27V CP53TEV)
MKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVT GTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVF TLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGL
SEQ ID NO:162(L27V CP54TEV)
atgatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaa ggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgc cgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccggg accctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccat caacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagc ctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttaca ctcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtc cagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaag
SEQ ID NO:163(L27V CP54TEV)
MIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTG TLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFT LEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLK
SEQ ID NO:164(L27V CP55TEV)
atggacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggt ggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccga acatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggacc ctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaa cggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagccta ctactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactc gaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccag tttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatc
SEQ ID NO:165(L27V CP55TEV)
MDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGT LWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTL EDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKI
SEQ ID NO:166(L27V CP56TEV)
Atgatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggt gtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaaca tgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctg tggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgg agtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctacta ctgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaa gatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagttt gtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcga
SEQ ID NO:167(L27V CP56TEV)
MIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTL WNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLE DFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKID
SEQ ID NO:168(L27V CP58TEV)
atggtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccc tgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcg actatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgac cggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgaga acttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttc gtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttca gaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccat
SEQ ID NO:169(L27V CP58TEV)
MVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWN GNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDF VGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIH
SEQ ID NO:170(L27V CP64TEV)
atgggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactt taaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgt atgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgac gagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcga gcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcg ataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgt aactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaa
SEQ ID NO:171(L27V CP64TEV)
MGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIID ERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQ TAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE
SEQ ID NO:172(L27V CP67TEV)
atgggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgat tctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggca tcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctg atcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcatttt ggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaa gttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggc tacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgat ccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagc
SEQ ID NO:173(L27V CP67TEV)
MGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERL INPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAG YNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLS
SEQ ID NO:174(L27V CP70TEV)
atgatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcacta tggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgt tcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc gacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaag ttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtg gaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctg gaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggat tgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcag
SEQ ID NO:175(L27V CP70TEV)
MMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINP DGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQ
SEQ ID NO:176(L27V CP73TEV)
atgatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacact ggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggca aaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctcc ctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtgg aggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagtt ctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgag cggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccag
SEQ ID NO:177(L27V CP73TEV)
MIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGS LLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQV LEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQ
SEQ ID NO:178(L27V CP76TEV)
atgatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcga cggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatca ctgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttc tggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaa tggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcag ggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaa cggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaa
SEQ ID NO:179(L27V CP76TEV)
MIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLF RVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQ GGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK
SEQ ID NO:180(L27V CP79TEV)
Atggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttac gccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccg ggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaacc atcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggaga gcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgttta cactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtg tccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaa gatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaag
SEQ ID NO:181(L27V CP79TEV)
MVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVT INGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGV SSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFK
SEQ ID NO:182(L27V CP80TEV)
atggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgcc gaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccggga ccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatc aacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcc tactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacac tcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtcc agtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagat cgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtg
SEQ ID NO:183(L27V CP80TEV)
MVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTI NGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVS SLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKV
SEQ ID NO:184(L27V CP81TEV)
atgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaa catgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccc tgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaac ggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctac tactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcg aagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagt ttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcga catccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtg
SEQ ID NO:185(L27V CP81TEV)
MYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN GVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSS LFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVV
SEQ ID NO:186(L27V CP82TEV)
atgcctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacat gatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgt ggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactac tgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaag atttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttg tttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacat ccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtac
SEQ ID NO:187(L27V CP82TEV)
MPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTING VTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSL FQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVY
SEQ ID NO:188(L27V CP83TEV)
atggtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgat cgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtgga acggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtg accggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactga gaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatt tcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgttt cagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatcca tgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccct
SEQ ID NO:189(L27V CP83TEV)
MVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV TGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLF QNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYP
SEQ ID NO:190(L27V CP84TEV)
atggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcga ctatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacg gcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaa cttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcg taggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcag aatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgt catcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg
SEQ ID NO:191(L27V CP84TEV)
MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT GWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQ NLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV
SEQ ID NO:192(L27V CP85TEV)
atggatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgacta tttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggca acaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggc tggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaactt gtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtag gggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaat ctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcat catcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggat
SEQ ID NO:193(L27V CP85TEV)
MDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTG WRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQN LGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVD
SEQ ID NO:194(L27V CP86TEV)
atgcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactattt cggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaaca aaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctgg cggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgta cttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtagggg actggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgat
SEQ ID NO:195(L27V CP86TEV)
MHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDD
SEQ ID NO:196(L27V CP87TEV)
atgcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgg acggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaa ttatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcgg ctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtactt ccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggact ggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggg gtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatccc gtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcat
SEQ ID NO:197(L27V CP87TEV)
MHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR LCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLG VSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDH
SEQ ID NO:198(L27V CP88TEV)
atgtttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacg gccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaatta tcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctg tgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttcca gagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggc ggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgta tgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac
SEQ ID NO:199(L27V CP88TEV)
MFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRL CERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGV SVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH
SEQ ID NO:200(L27V CP91TEV)
atgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatga aggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagc gcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataa cggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacag ccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaact ccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtct gagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg
SEQ ID NO:201(L27V CP91TEV)
MILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER ILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVT PIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKV
SEQ ID NO:202(L27V CP94TEV)
atgtatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgc cgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatca accccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg ggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttc tggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctaca acctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaa aggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcga tcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcacg
SEQ ID NO:203(L27V CP94TEV)
MYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA GSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQ RIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILH
SEQ ID NO:204(L27V CP95TEV)
atgggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgt gttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacc ccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggga agttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctgg tggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacc tggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaagg attgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatca gatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactat
SEQ IDNO:205(L27V CP95TEV)
MGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAG SSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHY
SEQ ID NO:206(L27V CP97TEV)
atgctggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcga cggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacg gctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttct ggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggagg aagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggacc aagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtc ctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatggg ccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcaca
SEQ ID NO:207(L27V CP97TEV)
MLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSS GGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIV LSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGT
SEQ ID NO:208(L27V CP100TEV)
Atggacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaa gatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgc tgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggagga agttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctgg tggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttg agcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggt gaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcga aaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatc
SEQ ID NO:209(L27V CP100TEV)
MDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGG SSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSG ENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVI
SEQ ID NO:210(L27V CP101TEV)
atgggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagat cactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgt tccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagt tctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtgg aatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagc agggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaa aacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaa aatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgac
SEQ ID NO:211(L27V CP101TEV)
MGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGS SGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGE NGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVID
SEQ ID NO:212(L27V CP102TEV)
atggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcac tgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttcc gcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttct ggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaat ggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagg gcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaac ggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaat ttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggg
SEQ ID NO:213(L27V CP102TEV)
MVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSS GGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGEN GLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDG
SEQ ID NO:214(L27V CP103TEV)
atgacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgt aaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcg taaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggt ggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggt gtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcg gtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggc ctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttt taaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggtt
SEQ ID NO:215(L27V CP103TEV)
MTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSG GEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENG LKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
SEQ ID NO:216(L27V CP104TEV)
atgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaac cgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaa ccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtgga gagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtt tacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtg tgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctg aagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaa ggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacg
SEQ ID NO:217(L27V CP104TEV)
MPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGG EPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGL KIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
SEQ ID NO:218(L27V CP105TEV)
atgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgg gaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaacca tcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagag cctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttac actcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgt ccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaag atcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggt ggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacg
SEQ ID NO:219(L27V CP105TEV)
MNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGE PTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLK IDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT
SEQ ID NO:220(L27V CP106TEV)
atgatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggac cctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatca acggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcct actactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacact cgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtcca gtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatc gacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggt gtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaac
SEQ ID NO:221(L27V CP106TEV)
MMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEP TTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKI DIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPN
SEQ ID NO:222(L27V CP109TEV)
atgtatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaa cggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtga ccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgag aacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagattt cgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttc agaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccat gtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgt ggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgac
SEQ ID NO:223(L27V CP109TEV)
MYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTE NLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIH VIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMID
SEQ ID NO:224(L27V CP112TEV)
atgcggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaa aattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggc ggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtac ttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtagggga ctggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcg gggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatca tcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga
SEQ ID NO:225(L27V CP112TEV)
MRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLY FQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVII PYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG
SEQ ID NO:226(L27V CP115TEV)
atggaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcga cgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcg agcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagc gataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggca gacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccg taactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaa ggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaa ggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtat
SEQ ID NO:227(L27V CP115TEV)
MEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQS DNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE GLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPY
SEQ ID NO:228(L27V CP120TEV)
atgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaa ccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgg gaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttct ggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaa cctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaa ggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgat cagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcacta tggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgtggcc
SEQ ID NO:229(L27V CP120TEV)
MFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSS GGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGD QMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIVA
SEQ ID NO:230(L27V CP121TEV)
atggacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccc cgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaa gttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggt ggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacct ggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaagga ttgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcag atgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatgg cacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttc
SEQ ID NO:231(L27V CP121TEV)
MDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSG GGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQ MGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVF
SEQ ID NO:232(L27V CP123TEV)
atgaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacgg ctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctg gtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggagga agttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggacca agtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcc tgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggc cagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacact ggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc
SEQ ID NO:233(L27V CP123TEV)
MKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGG SSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMG QIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDG
SEQ ID NO:234(L27V CP124TEV)
Atgaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctc cctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtg gaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagt tctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagt ccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctga gcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccag atcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggt aatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa
SEQ ID NO:235(L27V CP124TEV)
MKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGS SGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQ IEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGK
SEQ ID NO:236(L27V CP125TEV)
atgatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccct gctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggag gaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttct ggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtcct tgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcg gtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatc gaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaat cgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag
SEQ ID NO:237(L27V CP125TEV)
MITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSS GGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQI EKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKK
SEQ ID NO:238(L27V CP129TEV)
atggggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgt aaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtg gagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtg tttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcgg tgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcc tgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaattttt aaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttac gccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacc
SEQ ID NO:239(L27V CP129TEV)
MGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMV FTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIF KVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVT
SEQ ID NO:240(L27V CP130TEV)
atgaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaac catcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggag agcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgttt acactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgt gtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctga agatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaag gtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgcc gaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccggg
SEQ ID NO:241(L27V CP130TEV)
MTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVF TLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFK VVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTG
SEQ ID NO:242(L27V CP131TEV)
atgctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccat caacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagc ctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttaca ctcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtc cagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaaga tcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtg gtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaa catgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggacc
SEQ ID NO:243(L27V CP131TEV)
MLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFT LEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKV VYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGT
SEQ ID NO:244(L27V CP133TEV)
atgaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgg agtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctacta ctgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaa gatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagttt gtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgaca tccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtac cctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgat cgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggg
SEQ ID NO:245(L27V CP133TEV)
MNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLE DFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVY PVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLW
SEQ ID NO:246(L27V CP136TEV)
atgaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccgg ctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaact tgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgta ggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaa tctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtca tcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggat gatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactattt cggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaac
SEQ ID NO:247(L27V CP136TEV)
MKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFV GDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVD DHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGN
SEQ ID NO:248(L27V CP139TEV)
atggacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggct gtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttcc agagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactgg cggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggt gtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgt atgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggcc gtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcg acgagcgc
SEQ ID NO:249(L27V CP139TEV)
MDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDW RQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDER
SEQ ID NO:250(L27V CP140TEV)
atggagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtg cgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccaga gcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcgg cagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtc cgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatg aaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgta tgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgac
SEQ ID NO:251(L27V CP140TEV)
MERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWR QTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHF KVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIID
SEQ ID NO:252(L27V CP141TEV)
atgcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcga gcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcg ataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgt aactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaag gtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatga aggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgag
SEQ ID NO:253(L27V CP141TEV)
MRLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQ TAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDE
SEQ ID NO:254(L27V CP142TEV)
atgctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcg cattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgata acggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagaca gccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaac tccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtc tgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaagg catcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgc
SEQ ID NO:255(L27V CP142TEV)
MLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQT AGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKV ILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDER
SEQ ID NO:256(L27V CP143TEV)
atgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcat tttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacg gaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagcc ggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactcc gatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctga gcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatt ctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcat cgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctg
SEQ ID NO:257(L27V CP143TEV)
MINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTA GYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVI LHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERL
SEQ ID NO:258(L27V CP144TEV)
atgaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcatttt ggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaa gttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggc tacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgat ccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcg gcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctg cactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgc cgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatc
SEQ ID NO:259(L27V CP144TEV)
MNPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAG YNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVIL HYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLI
SEQ ID NO:260(L27V CP145TEV)
atgcccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggc gggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagtt ctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctac aacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatcca aaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcg atcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcac tatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgt gttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaac
SEQ ID NO:261(L27V CP145TEV)
MPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGY NLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILH YGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLIN
SEQ ID NO:262(L27V CP146TEV)
atggacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggg aagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctg gtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaac ctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaag gattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatc agatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactat ggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgtt cgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc
SEQ ID NO:263(L27V CP146TEV)
MDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYN LDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHY GTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINP
SEQ ID NO:264(L27V CP147TEV)
atgggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaag ttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtg gaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctg gaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggat tgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcaga tgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggc acactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcga cggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgac
SEQ ID NO:265(L27V CP147TEV)
MGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYG TLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPD
SEQ ID NO:266(L27V CP148TEV)
atgtccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttc tggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggag gaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggac caagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgt cctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgg gccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcaca ctggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacgg caaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggc
SEQ ID NO:267(L27V CP148TEV)
MSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLD QVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGT LVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDG
SEQ ID NO:268(L27V CP149TEV)
atgctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctgg tggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaa gttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaa gtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcct gagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggcc agatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactg gtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaa aaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctcc
SEQ ID NO:269(L27V CP149TEV)
MLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQ VLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTL VIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGS
SEQ ID NO:270(L27V CP150TEV)
atgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtgg aggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagtt ctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgag cggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccaga tcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggta atcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaa gatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctg
SEQ ID NO:271(L27V CP150TEV)
MLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQV LEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLV IDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSL
SEQ ID NO:272(L27V CP151TEV)
atgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggagg aagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctg gtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtcctt gagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcgg tgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcg aaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatc gacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagat cactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctg
SEQ ID NO:273(L27V CP151TEV)
MFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVL EQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVI DGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLL
SEQ ID NO:274(L27V CP154TEV)
atgaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctgg tggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatgg tgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggc ggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacgg cctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaattt ttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggtt acgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaac cgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgta
SEQ ID NO:275(L27V CP154TEV)
MTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQG GVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRV
SEQ ID NO:276(L27V CP156TEV)
atgaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggaga gcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgttta cactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtg tccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaa gatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaagg tggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccg aacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggac cctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatc
SEQ ID NO:277(L27V CP156TEV)
MNGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGV SSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTP NMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTI
SEQ ID NO:278(L27V CP157TEV)
atgggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcc tactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacac tcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtcc agtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagat cgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtgg tgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccct gtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaac
SEQ ID NO:279(L27V CP157TEV)
MGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVS SLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN
SEQ ID NO:280(L27V CP158TEV)
atggtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctac tactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcg aagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagt ttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcga catccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgt accctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatg atcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtg gaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga
SEQ ID NO:281(L27V CP158TEV)
MVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSS LFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNM IDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTING
SEQ ID NO:282(L27V CP160TEV)
atgggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactga gaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatt tcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgttt cagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatcca tgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctg tggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgac tatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacgg caacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc
SEQ ID NO:283(L27V CP160TEV)
MGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLF QNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMID YFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT
SEQ ID NO:284(L27V CP163TEV)
atgctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgta cttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtagggg actggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcgg
SEQ ID NO:285(L27V CP163TEV)
MLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
SEQ ID NO:286(L27V CP166TEV)
Atgcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagag cgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggc agacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtcc gtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatga aggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttta aggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtat gaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacga gcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgag
SEQ ID NO:287(L27V CP166TEV)
MRILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVS VTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPY EGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCE
SEQ ID NO:288(pCA9FKBP-L27V02A157-169)gagtgcaggtggaaaccatctccccaggaga cgggcgcaccttccccaagcgcggccagacctgcgtggtgcactacaccgggatgcttgaagatggaaagaaatttg attcctcccgggacagaaacaagccctttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggg gttgcccagatgagtgtgggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcaccc aggcatcatcccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtg gaggcagcggtggagtgaccggctggcggctgtgcgaacgcattctggcg
SEQ ID NO:289(pCA9FKBP-L27V02A157-169)
GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTIS PDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGGVTGWRLCERILA
SEQ ID NO:290(pCA10L27V02A1-156-FRB)
atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccga gtaaccatcaacggagtgaccggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtg gcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagc ccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagattta atggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctcta ttatcatgtgttccgacgaatctca
SEQ ID NO:291(pCA10L27V02A1-156-FRB)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDL MEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:292(pCA26FKBP-L27V02A1-156)
ggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgcacta caccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctaggca agcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactatatct ccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtggagct tctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttggggact ggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggg gtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatccc gtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatc actttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacgg ccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattat cgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaac
SEQ ID NO:293(pCA26FKBP-L27V02A1-156)
GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTIS PDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLG VSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGR PYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN
SEQ ID NO:294(pCA25L27V02A157-169-FRB)
atgggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggc catcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaag gcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaat caggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacct cacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:295(pCA25L27V02A157-169-FRB)
MGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN QAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:296(pCA3FKBP-L27V02A103-169)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtacgccgaacatgatcgactatttcggacggc cgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatc gacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtg cgaacgcattctggc
SEQ ID NO:297(pCA3FKBP-L27V02A103-169)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKII DERLINPDGSLLFRVTINGVTGWRLCERIL
SEQ ID NO:298(pCA4L27V02A1-102-FRB)
atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaag aggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatg gaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtg gtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgac gaatctca
SEQ ID NO:299(pCA4L27V02A1-102-FRB)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKII DERLINPDGSLLFRVTINGVTGWRLCERIL
SEQ ID NO:300(pCA19L27V02A103-169-FRB)
atgacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgt aacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgag taaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagc tccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaa cgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacat cctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtc aaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:301(pCA19L27V02A103-169-FRB)
MTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGS SVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNV KDLTQAWDLYYHVFRRIS
SEQ ID NO:302(pCA20FKBP-L27V02A1-102)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggtt
SEQ ID NO:303(pCA20FKBP-L27V02A1-102)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
SEQ ID NO:304(pCA11FKBP-L27V02A84-169)atgggagtgcaggtggaaaccatctccccag gagacgggcgcaccttccccaagcgcggccagacctgcgtggtgcactacaccgggatgcttgaagatggaaagaaa tttgattcctcccgggacagaaacaagccctttaagtttatgctaggcaagcaggaggtgatccgaggctgggaaga aggggttgcccagatgagtgtgggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggc acccaggcatcatcccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctcc ggtggaggcagcggtgatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgcc gaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacaggga ccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatc aacggagtgaccggctggcggctgtgcgaacgcattctggcg
SEQ ID NO:305(pCA11FKBP-L27V02A84-169)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAV FDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:306(pCA12L27V02A1-83-FRB)
atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagat gtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctgg agcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagat ttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacct ctattatcatgtgttccgacgaatctca
SEQ ID NO:307(pCA12L27V02A1-83-FRB)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRD LMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:308 (pCA14L27V02A1-83 (fusion))
atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtg
SEQ ID NO:309 (pCA14L27V02A1-83 (fusion))
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPV
SEQ ID NO:310 (pCA13L27V02A84-169 (fusion))
atggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcga ctatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacg gcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgacc ggctggcggctgtgcgaacgcattctggcg
SEQ ID NO:311 (pCA13L27V02A84-169 (fusion))
MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT GWRLCERILA
SEQ ID NO:312(pCA28FKBP1-83)atgggagtgcaggtggaaaccatctccccaggagacgggc gcaccttccccaagcgcggccagacctgcgtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcc tcccgggacagaaacaagccctttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgc ccagatgagtgtgggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggca tcatcccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtggaggc agcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtcct tgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcg gtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatc gaaaaaatttttaaggtggtgtaccctgtg
SEQ ID NO:313(pCA28FKBP1-83)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV
SEQ ID NO:314(pCA27L27V02A84-169FRB)atggatgatcatcactttaaggtgatcctgcac tatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgt gttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacc ccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggga ggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatc tcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggg gcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcagg aagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:315(pCA27L27V02A84-169FRB)
MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT GWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYG RDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:316(HT7-Keap1)
atggcagaaatcggtactggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcactacgtcga tgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacctcctcctacgtgtggcgcaacatca tcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtatgggcaaatccgacaaaccagacctg ggttatttcttcgacgaccacgtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggt cattcacgactggggctccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcattta tggagttcatccgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcacc accgacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgccc gctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcc caaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcaccag tcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccggccgaagccgctcgcctggccaa aagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctgcaagaagacaacccggacctgatcg gcagcgagatcgcgcgctggctgtcgacgctcgagatttccggcgagccaaccactgaggatctgtactttcagagc gataacgcgatcgctttcgaaggagatagaaccatgcagccagatcccaggcctagcggggctggggcctgctgccg attcctgcccctgcagtcacagtgccctgagggggcaggggacgcggtgatgtacgcctccactgagtgcaaggcgg aggtgacgccctcccagcatggcaaccgcaccttcagctacaccctggaggatcataccaagcaggcctttggcatc atgaacgagctgcggctcagccagcagctgtgtgacgtcacactgcaggtcaagtaccaggatgcaccggccgccca gttcatggcccacaaggtggtgctggcctcatccagccctgttttcaaggccatgttcaccaacgggctgcgggagc agggcatggaggtggtgtccattgagggtatccaccccaaggtcatggagcgcctcattgaattcgcctacacggcc tccatctccatgggcgagaagtgtgtcctccacgtcatgaacggcgctgtcatgtaccagatcgacagcgttgtccg tgcctgcagtgacttcctggtgcagcagctggaccccagcaatgccatcggcatcgccaacttcgctgagcagattg gctgtgtggagttgcaccagcgtgcccgggagtacatctacatgcattttggggaggtggccaagcaagaggagttc ttcaacctgtcccactgccaactggtgaccctcatcagccgggacgacctgaacgtgcgctgcgagtccgaggtctt ccacgcctgcatcaactgggtcaagtacgactgcgaacagcgacggttctacgtccaggcgctgctgcgggccgtgc gctgccactcgttgacgccgaacttcctgcagatgcagctgcagaagtgcgagatcctgcagtccgactcccgctgc aaggactacctggtcaagatcttcgaggagctcaccctgcacaagcccacgcaggtgatgccctgccgggcgcccaa ggtgggccgcctgatctacaccgcgggcggctacttccgacagtcgctcagctacctggaggcttacaaccccagta acggcacctggctccggttggcggacctgcaggtgccgcggagcggcctggccggctgcgtggtgggcgggctgttg tacgccgtgggcggcaggaacaactcgcccgacggcaacaccgactccagcgccctggactgttacaaccccatgac caatcagtggtcgccctgcgcccccatgagcgtgccccgtaaccgcatcggggtgggggtcatcgatggccacatct atgccgtcggcggctcccacggctgcatccaccacaacagtgtggagaggtatgagccagagcgggatgagtggcac ttggtggccccaatgctgacacgaaggatcggggtgggcgtggctgtcctcaatcgtctgctttatgccgtgggggg ctttgacgggacaaaccgccttaattcagctgagtgttactacccagagaggaacgagtggcgaatgatcacagcaa tgaacaccatccgaagcggggcaggcgtctgcgtcctgcacaactgtatctatgctgctgggggctatgatggtcag gaccagctgaacagcgtggagcgctacgatgtggaaacagagacgtggactttcgtagcccccatgaagcaccggcg aagtgccctggggatcactgtccaccaggggagaatctacgtccttggaggctatgatggtcacacgttcctggaca gtgtggagtgttacgacccagatacagacacctggagcgaggtgacccgaatgacatcgggccggagtggggtgggc gtggctgtcaccatggagccctgccggaagcagattgaccagcagaactgtacctgttacgtagtt
SEQ ID NO:317(Nrf2)
TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGT TGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACT AGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGT AAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGC CAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTAT CATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTT ACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACA CCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTT TGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGT ACGGTGGGAGGTCTATATAAGCAGAGCTgGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGCGGTAGTTTATC ACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGC ACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAG ACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCC AGTTCAATTACAGCTCTTAAGGCTAGAGTATTAATACGACTCACTATAGGGCTAGCGATCGCCATGATGGACTTGGA GCTGCCGCCGCCGGGACTCCCGTCCCAGCAGGACATGGATTTGATTGACATACTTTGGAGGCAAGATATAGATCTTG GAGTAAGTCGAGAAGTATTTGACTTCAGTCAGCGACGGAAAGAGTATGAGCTGGAAAAACAGAAAAAACTTGAAAAG GAAAGACAAGAACAACTCCAAAAGGAGCAAGAGAAAGCCTTTTTCGCTCAGTTACAACTAGATGAAGAGACAGGTGA ATTTCTCCCAATTCAGCCAGCCCAGCACATCCAGTCAGAAACCAGTTCTCTCggtggttcaggtggtggcgggagcg gtggaGGGAGCagCGGTGGAgtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctg gaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggat tgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcaga tgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggc acactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcga cggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacg gctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgtaaggccgc gacTCTAGAGTCGACCTGCAGGCATGCAAGCTgatcCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCT GCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGT TTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTA ACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTG GGAGGTTTTTTtAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCgaattTTAACAAAATATTAACGCTTACAATT TCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGCGGATCTGCGCAGCACCATGGC CTGAAATAACCTCTGAAAGAGGAACTTGGTTAGGTACCTTCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAG TTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAG GTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCC CGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTT TTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCT AGGCTTTTGCAAAAAGCTTaattaactgttgacaattaatcatcggcatagtatatcggcatagtataatacgacaa ggtgaggaactaaacccaggaggcagatcatgattgaacaagatggattgcacgcaggttctcCGGCCGCTTGGGTG GAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCA GGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTAT CGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTA TTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGC AATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCAC GTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTG TTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATAT CATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAG CGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCC GCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATG ACCGACCAAGCGACGCCCAACCTGCCATCACGATGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTG GTTTTTTGTGTGAATCGATAGCGATAAggatccTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTG ACATTCATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACCCGGTATCAGCTCACTCAAAGGCGGTAATACG GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAA AGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGAC CCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGT ATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCC TTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGA CAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAA ACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATTTCAAGAAGATCC TTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAA AAAGGATCTTCACCTAGATCCTTTTATAGTccggaaatacaggaacgcacgctggatggcccttcgctgggatggtg aaaccatgaaaaatggcagcttcagtggattaagtgggggtaatgtggcctgtaccctctggttgcataggtattca tacggttaaaatttatcaggcgcgattgcggcagtttttcgggtggtttgttgccatttttacctgtctgctgccgt gatcgcgctgaacgcgttttagcggtgcgtacaattaagggattatggtaaatccacttactgtctgccctcgtagc catcgagataaaccgcagtACTCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCT
SEQ ID NO:318(L27V02B)
ATGGTCTTCACACTCGAAGATTTCGTAGGTGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACA GGGTGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAA ATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGG GGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTG TAACTGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGA GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCG
SEQ ID NO:319(L27V01)
ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACA GGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAA ACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGATCAAATGGGCCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGG GGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTG TAACCGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGC GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCG
SEQ ID NO:320(L27V01-PEST00)
ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACA GGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAA ACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGATCAAATGGGCCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGG GGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTG TAACCGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGC GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCGAATTCACACGGCTTTCCGCCCGAGGT TGAAGAGCAAGCCGCCGGTACATTGCCTATGTCCTGCGCACAAGAAAGCGGTATGGACCGGCACCCAGCCGCTTGTG CTTCAGCTCGCATCAACGTC
SEQ ID NO:321(IL601-L27V01)
ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGCCTGCTCCTGGTGTTGCCTGCTGCCTT CCCTGCCCCAGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTCC TTGAACAGGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGC GGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGATCAAATGGGCCAGAT CGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAA TCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAG ATCACTGTAACCGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCT GTTCCGCGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCG
SEQ ID NO:322(L27V02A)
ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACA GGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAA ATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGG GGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTG TAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGA GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCG
SEQ ID NO:323(L27V02A-PEST01)
ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACA GGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAA ATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGG GGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTG TAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGA GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCGAATTCTCACGGCTTTCCGCCTGAGGT TGAAGAGCAAGCCGCCGGTACATTGCCTATGTCCTGCGCACAAGAAAGCGGTATGGACCGGCACCCAGCCGCTTGTG CTTCAGCTCGCATCAACGTC
SEQ ID NO:324(IL601-L27V02A)
ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGCCTGCTCCTGGTGTTGCCTGCTGCCTT CCCTGCCCCAGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCC TTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGC GGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGAT CGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAA TCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAG ATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCT GTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCG
SEQ ID NO:325(L27V03)
ATGGTCTTCACACTCGAAGATTTCGTAGGTGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTTCTTGAACA GGGTGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCAATCCAGAGGATAGTCCTGAGTGGTGAAA ATGGGCTGAAGATCGACATCCATGTCATCATCCCTTATGAAGGTCTGAGCGGCGATCAGATGGGGCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGG TGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATTACTG TCACTGGAACCCTGTGGAATGGGAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCACTGCTGTTCCGA GTAACCATCAATGGTGTTACCGGCTGGCGGCTCTGCGAACGCATTCTAGCA
SEQ ID NO:326(L27V03-PEST02)
ATGGTCTTCACACTCGAAGATTTCGTAGGTGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTTCTTGAACA GGGTGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCAATCCAGAGGATAGTCCTGAGTGGTGAAA ATGGGCTGAAGATCGACATCCATGTCATCATCCCTTATGAAGGTCTGAGCGGCGATCAGATGGGGCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGG TGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATTACTG TCACTGGAACCCTGTGGAATGGGAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCACTGCTGTTCCGA GTAACCATCAATGGTGTTACCGGCTGGCGGCTCTGCGAACGCATTCTAGCAAATAGTCACGGCTTTCCGCCTGAGGT TGAAGAGCAAGCCGCCGGTACATTGCCTATGTCCTGCGCACAAGAAAGCGGTATGGACCGGCACCCAGCCGCTTGTG CTTCAGCTCGCATCAACGTC
SEQ ID NO:327(IL602-L27V03)
ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTCGCCTTCTCCCTGGGCCTGCTCCTGGTGTTGCCCGCTGCCTT TCCTGCCCCAGTCTTCACACTCGAAGATTTCGTAGGTGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTTC TTGAACAGGGTGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCAATCCAGAGGATAGTCCTGAGT GGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCTTATGAAGGTCTGAGCGGCGATCAGATGGGGCAGAT CGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAA TCGACGGTGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAG ATTACTGTCACTGGAACCCTGTGGAATGGGAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCACTGCT GTTCCGAGTAACCATCAATGGTGTTACCGGCTGG
SEQ ID NO:328 (joint)
GSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGG
SEQ ID NO:329 (FABP consensus sequence)
[GSAIVK]-{FE}-[FYW]-x-[LIVMF]-x-x-{K}-x-[NHG]-[FY]-[DE]-x-[LIVMFY]- [LIVM]-{N}-{G}-[LIVMAKR]
SEQ ID NO:330 (OGLUC consensus sequence)
[GSAIVK]-{FE}-[FYW]-x-[LIVMFSYQ]-x-x-{K}-x-[NHGK]-x-[DE]-x-[LIVMFY]- [LIVMWF]-x-{G}-[LIVMAKRG]
SEQ ID NO:331(9B8PCA1(pF5A/Met-[9B8opt(51-169)]-GGGGSGGGSS-FRB)
Atgggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggctatcagatgggccagatcgaaaa aatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacg gggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcact gtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccg cgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaggaggtggctcaggtggaggga gctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaagg aacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaac atcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatg tcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:332(9B8PCA1(pF5A/Met-[9B8opt(51-169)]-GGGGSGGGSS-FRB)
MGLKIDIHVIIPYEGLSGYQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKIT VTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGER NVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:333(9B8PCA2(pF5A/[9B8opt(1-50)]-GGGGSGGGSS-FRB)
Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagca gggcggtctgtccagtttgtttcagaaactcggggtgtccgtaacaccgatccaaaagattgtcctgagcggtgaaa acggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagag gcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatgga acggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggt gcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacga atctca
SEQ ID NO:334(9B8PCA2(pF5A/[9B8opt(1-50)]-GGGGSGGGSS-FRB)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGGGGSGGGSSVAILWHEMWHEGLEE ASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRR IS
SEQ ID NO:335(9B8PCA3(&pF5A/FKBP-GGGSSGGGSG-[9B8opt(51-169)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtggcctgaagatcgacatccatgtcatcatcc cgtatgaaggtctgagcggctatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcat cactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacg gccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaatta tcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctg tgcgagcgcattttggcg
SEQ ID NO:336(9B8PCA3(&pF5A/FKBP-GGGSSGGGSG-[9B8opt(51-169)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGGLKIDIHVIIPYEGLSGYQMGQIEKIFKVVYPVDDH HFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRL CERILA
SEQ ID NO:337(9B8PCA4(pF5A/FKBP-GGGSSGGGSG-[9B8opt(1-50)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtgtttacactcgaagatttcgtagggg actggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtctgtccagtttgtttcagaaactc ggggtgtccgtaacaccgatccaaaagattgtcctgagcggtgaaaac
SEQ ID NO:338(9B8PCA4(pF5A/FKBP-GGGSSGGGSG-[9B8opt(1-50)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKL GVSVTPIQKIVLSGEN
SEQ ID NO:339(K33N+L27V+K43R+Y68D)
Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagca gggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaacaccgatccaaaggattgtcctgagcggtgaaa acggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgc gtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg
SEQ ID NO:340(K33N+L27V+K43R+Y68D)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:341(K33N+L27V+T39T+K43R+S66N)
Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagca gggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa acggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgaacggctatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgc gtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg
SEQ ID NO:342(K33N+L27V+T39T+K43R+S66N)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLNGYQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILA
SEQ ID NO:343(pCA31pCA L27V02A45-169FRB)
atggtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgacca aatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatg gcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttc gacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccga cggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggag gtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgt ttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggccc ccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagt acatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:344(pCA31pCA L27V02A45-169FRB)
MVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVF DGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASR LYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:345(pCA32FKBP L27V02A1-44)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggatt
SEQ ID NO:346(pCA32FKBP L27V02A1-44)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRI
SEQ ID NO:347(pCA33pCA L27V02A46-169FRB)
atgctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaat gggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggca cactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgac ggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacgg ctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtg gctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttg tactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggccccca gactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtaca tgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:348(pCA33pCA L27V02A46-169FRB)
MLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFD GKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRL YFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:349(pCA34pCA FKBP1-45L27V02A)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtc
SEQ ID NO:350(pCA34pCA FKBP1-45L27V02A)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIV
SEQ ID NO:351(pCA35pCA L27V02A100-169FRB)
atggacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaa gatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgc tgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggt ggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttgg ggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctga aggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatca gggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:352(pCA35pCA L27V02A100-169FRB)
MDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSG GGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKS GNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:353(pCA36FKBP L27V02A1-99)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatc
SEQ ID NO:354(pCA36FKBP L27V02A1-99)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVI
SEQ ID NO:355(pCA37L27V02A101-169FRB)
atgggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagat cactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgt tccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtgga gggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttgggga aaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaagg aaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcaggg aatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:356(pCA37L27V02A101-169FRB)
MGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGG GSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSG NVKDLTQAWDLYYHVFRRIS
SEQ ID NO:357(pCA38FKBP1-100L27V02A)
Atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgac
SEQ ID NO:358(pCA38FKBP1-100L27V02A)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVID
SEQ ID NO:359(pCA39L27V02A102-169FRB)
Atggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcac tgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttcc gagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggaggg agctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaag gaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaa catcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaat gtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:360(pCA39L27V02A102-169FRB)
MVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGG SSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGN VKDLTQAWDLYYHVFRRIS
SEQ ID NO:361(pCA40FKBP L27V02A1-101)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggg
SEQ ID NO:362(pCA40FKBP L27V02A1-101)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDG
SEQ ID NO:363(pCA41L27V02A143-169FRB)
atgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcat tctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctgg aagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatg atggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaaga gtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttcc gacgaatctca
SEQ ID NO:364(pCA41L27V02A143-169FRB)
MINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAM MERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:365(pCA42FKBP1-142L27V02A)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctg
SEQ ID NO:366(pCA42FKBP1-142L27V02A)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERL
SEQ ID NO:367(pCA43L27V02A145-169FRB)
atgcccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggc gggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagagg catctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaa cggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtg caggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaa tctca
SEQ ID NO:368(pCA43L27V02A145-169FRB)
MPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMME RGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:369(pCA44FKBP1-144L27V02A)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaac
SEQ ID NO:370(pCA44FKBP1-144L27V02A)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLIN
SEQ ID NO:371(pCA45L27V02A147-169FRB)
atgggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggagg aggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctc gtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggc ccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaa gtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:372(pCA45L27V02A147-169FRB)
MGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERG PQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:373(pCA46FKBP-L27V02A1-146)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagcactttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgac
SEQ ID NO:374(pCA46L27V02A-FKBP1-146)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKHFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPD
SEQ ID NO:375(pCA47L27V02A148-169FRB)
atgtccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggagg tggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtt tgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccc cagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagta catgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:376(pCA47L27V02A148-169FRB)
MSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGP QTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:377(pCA48FKBP-L27V02A1-147)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggc
SEQ ID NO:378(pCA48FKBP-L27V02A1-147)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDG
SEQ ID NO:379(pCA49L27V02A156-169FRB)
atgaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgt ggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtga aaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatccttt aatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaagga cctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:380(pCA49L27V02A156-169FRB)
MNGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSF NQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:381(pCA50FKBP-L27V02A1-155)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatc
SEQ ID NO:382(pCA50FKBP-L27V02A1-155)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTI
SEQ ID NO:383(pCA51L27V02A158-169FRB)
atggtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccat cctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggca tgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcag gcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcac ccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:384(pCA51L27V02A158-169FRB)
MVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQ AYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:385(pCA52FKBP1-157L27V02A)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacgga
SEQ ID NO:386(pCA52FKBP1-157L27V02A)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTING
SEQ ID NO:387(pCA53L27V02A166-169FRB)
atgcgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatga aggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgc atgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggag gcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatca tgtgttccgacgaatctca
SEQ ID NO:388(pCA53L27V02A166-169FRB)
MRILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLME AQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:389(pCA54FKBP L27V02A1-165)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggc tgtgcgaa
SEQ ID NO:390(pCA54FKBP L27V02A1-165)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCE
SEQ ID NO:391(pCA55FKBP L27V02A1-47)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagc
SEQ ID NO:392(pCA55FKBP L27V02A1-47)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLS
SEQ ID NO:393(pCA56L27V02A48-169-FRB)
Atgggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggcca gatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactgg taatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa aagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccct gctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcag gtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtacttt ggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactct gaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaat cagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:394(pCA56L27V02A48-169-FRB)
MGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGK KITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYF GERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:395(pCA57FKBP L27V02A1-49)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaa
SEQ ID NO:396(pCA57FKBP L27V02A1-49)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGE
SEQ ID NO:397(pCA58pCA L27V02A50-169FRB)
atgaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcga aaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcg acggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatc actgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgtt ccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggag ggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaa aggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaagga aacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcaggga atgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:398(pCA58pCA L27V02A50-169FRB)
MNGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKI TVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGE RNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:399(pCA59FKBP L27V02A1-82)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct
SEQ ID NO:400(pCA59FKBP L27V02A1-82)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYP
SEQ ID NO:401(pCA60L27V02A83-169-FRB)
atggtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgat cgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtgga acggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtg accggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctg gcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttg aggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctat ggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagc ctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:402(pCA60L27V02A83-16-FRB)
MVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV TGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAY GRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:403(pCA61FKBP L27V02A1-84)
Atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggat
SEQ ID NO:404(pCA61FKBP L27V02A1-84)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVD
SEQ ID NO:405(pCA62L27V02A85-169-FRB)
atggatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgacta tttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggca acaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggc tggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatga gatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgc tggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcga gatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctggga cctctattatcatgtgttccgacgaatctca
SEQ ID NO:406(pCA62L27V02A85-169-FRB)
MDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTG WRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR DLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:407(pCA63FKBP L27V02A1-122)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggc
SEQ ID NO:408(pCA63FKBP L27V02A1-122)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDG
SEQ ID NO:409(pCA64L27V02A123-169-FRB)
atgaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacgg ctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtg gctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttg tactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggccccca gactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtaca tgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:410(pCA64L27V02A123-169-FRB)
MKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRL YFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:411(pCA65FKBP L27V02A1-123)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaa
SEQ ID NO:412(pCA65FKBP L27V02A1-123)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGK
SEQ ID NO:413(pCA66L27V02A124-169FRB)
atgaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctc cctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggct caggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtac tttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagac tctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatga aatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:414(pCA66L27V02A124-169FRB)
MKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLY FGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:415(pCA67L27V02A1-168)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggc tgtgcgaacgcattctg
SEQ ID NO:416(pCA67L27V02A1-168)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggc tgtgcgaacgcattctg
SEQ ID NO:417(pCA67L27V02A1-168)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERIL
SEQ ID NO:418 (* pCA68L27V02A169FRB)
Atggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctgga agaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatga tggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagag tggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccg acgaatctca
SEQ ID NO:419 (* pCA68L27V02A169FRB)
MAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQE WCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:420(pCA69FKBP L27V02A1-166)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggc tgtgcgaacgc
SEQ ID NO:421(pCA69FKBP L27V02A1-166)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER
SEQ ID NO:422 (* pCA70L27V02A167-169FRB)
Atgattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaagg cctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatg ctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcc caagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgt gttccgacgaatctca
SEQ ID NO:423 (* pCA70L27V02A167-169FRB)
MILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:424(pCA71FKBP L27V02A1-164)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggc tgtgc
SEQ ID NO:425(pCA71FKBP L27V02A1-164)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLC
SEQ ID NO:426(pCA72L27V02A165-169FRB)
atggaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggca tgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagccct tgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatg gaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctatta tcatgtgttccgacgaatctca
SEQ ID NO:427 (mode sequences)
GSAIVK
SEQ ID NO:428(pCA72L27V02A165-169FRB)
MERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLM EAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:429(pCA73FKBP L27V02A1-162)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcgg
SEQ ID NO:430(pCA73FKBP L27V02A1-162)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR
SEQ ID NO:431(pCA74L27V02A163-169FRB)
atgctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagat gtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctgg agcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagat ttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacct ctattatcatgtgttccgacgaatctca
SEQ ID NO:432(pCA74L27V02A163-169FRB)
MLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRD LMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:433(pCA75FKBP L27V02A1-160)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggc
SEQ ID NO:434(pCA75FKBP L27V02A1-160)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTG
SEQ ID NO:435(pCA76L27V02A161-169FRB)
atgtggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggca tgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgagg tgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggt cgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctg ggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:436(pCA76L27V02A161-169FRB)
MWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYG RDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:437(pCA77FKBP L27V02A1-158)
atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtggtgca ctacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccctttaagtttatgctag gcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtcagagagccaaactgactata tctccagattatgcctatggtgccactgggcacccaggcatcatcccaccacatgccactctcgtcttcgatgtgga gcttctaaaactggaaggaggagggagctccggtggaggcagcggtatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtg
SEQ ID NO:438(pCA77FKBP L27V02A1-158)
MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTI SPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV
SEQ ID NO:439(pCA78L27V02A159-169FRB)
Atgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcct ctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgt ttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcc tatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcaccca agcctgggacctctattatcatgtgttccgacgaatctca
SEQ ID NO:440(pCA78L27V02A159-169FRB)
MTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQA YGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS
SEQ ID NO:441RIIbB (nucleotide sequence)
ATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGT GATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTG AATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCT CGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCAT TGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAA GGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTA
SEQ ID NO:442RIIbB (aminoacid sequence)
MYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIA RCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIV
SEQ ID NO:443(L27V CP13TEV)
atgacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtc cgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatg aaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgta tgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacg agcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgag cgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcga taacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcag
SEQ ID NO:444(L27V CP13TEV)
MTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHF KVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCE RILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQ
SEQ ID NO:445(L27V CP57TEV)
Atgcatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgta ccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatga tcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtgg aacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagt gaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactg agaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagat ttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtt tcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatc
SEQ ID NO:446(L27V CP57TEV)
MHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLW NGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLED FVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDI
SEQ ID NO:447(L27V CP98TEV)
atggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacgg caaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggct ccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggt ggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaag ttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaag tccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctg agcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggcca gatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactg
SEQ ID NO:448(L27V CP98TEV)
MVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSG GGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVL SGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTL
SEQ ID NO:449 (human kinase protein C α (3KCD) (nucleotide sequence)
ATGGCTGACGTTTTCCCGGGCAACGACTCCACGGCGTCTCAGGACGTGGCCAACCGCTTCGCCCGCAAAGGGGCGCT GAGGCAGAAGAACGTGCACGAGGTGAAGGACCACAAATTCATCGCGCGCTTCTTCAAGCAGCCCACCTTCTGCAGCC ACTGCACCGACTTCATCTGGGGGTTTGGGAAACAAGGCTTCCAGTGCCAAGTTTGCTGTTTTGTGGTCCACAAGAGG TGCCATGAATTTGTTACTTTTTCTTGTCCGGGTGCGGATAAGGGACCCGACACTGATGACCCCAGGAGCAAGCACAA GTTCAAAATCCACACTTACGGAAGCCCCACCTTCTGCGATCACTGTGGGTCACTGCTCTATGGACTTATCCATCAAG GGATGAAATGTGACACCTGCGATATGAACGTTCACAAGCAATGCGTCATCAATGTCCCCAGCCTCTGCGGAATGGAT CACACTGAGAAGAGGGGGCGGATTTACCTAAAGGCTGAGGTTGCTGATGAAAAGCTCCATGTCACAGTACGAGATGC AAAAAATCTAATCCCTATGGATCCAAACGGGCTTTCAGATCCTTATGTGAAGCTGAAACTTATTCCTGATCCCAAGA ATGAAAGCAAGCAAAAAACCAAAACCATCCGCTCCACACTAAATCCGCAGTGGAATGAGTCCTTTACATTCAAATTG AAACCTTCAGACAAAGACCGACGACTGTCTGTAGAAATCTGGGACTGGGATCGAACAACAAGGAATGACTTCATGGG ATCCCTTTCCTTTGGAGTTTCGGAGCTGATGAAGATGCCGGCCAGTGGATGGTACAAGTTgCTTAACCAAGAAGAAG GTGAGTACTACAACGTACCCATTCCGGAAGGGGACGAGGAAGGAAACATGGAACTCAGGCAGAAATTCGAGAAAGCC AAACTTGGCCCTGCTGGCAACAAAGTCATCAGTCCCTCTGAAGACAGGAAACAACCTTCCAACAACCTTGACCGAGT GAAACTCACGGACTTCAATTTCCTCATGGTGTTGGGAAAGGGGAGTTTTGGAAAGGTGATGCTTGCCGACAGGAAGG GCACAGAAGAACTGTATGCAATCAAAATCCTGAAGAAGGATGTGGTGATTCAGGATGATGACGTGGAGTGCACCATG GTAGAAAAGCGAGTCTTGGCCCTGCTTGACAAACCCCCGTTCTTGACGCAGCTGCACTCCTGCTTCCAGACAGTGGA TCGGCTGTACTTCGTCATGGAATATGTCAACGGTGGGGACCTCATGTACCACATTCAGCAAGTAGGAAAATTTAAGG AACCACAAGCAGTATTCTATGCGGCAGAGATTTCCATCGGATTGTTCTTTCTTCATAAAAGAGGAATCATTTATAGG GATCTGAAGTTAGATAACGTCATGTTGGATTCAGAAGGACATATCAAAATTGCTGACTTTGGGATGTGCAAGGAACA CATGATGGATGGAGTCACGACCAGGACCTTCTGTGGGACTCCAGATTATATCGCCCCAGAGATAATCGCTTATCAGC CGTATGGAAAATCTGTGGACTGGTGGGCCTATGGCGTCCTGTTGTATGAAATGCTTGCCGGGCAGCCTCCATTTGAT GGTGAAGATGAAGACGAGCTATTTCAGTCTATCATGGAGCACAACGTTTCCTATCCAAAATCCTTGTCCAAGGAGGC TGTTTCTATCTGCAAAGGACTGATGACCAAACACCCAGCCAAGCGGCTGGGCTGTGGGCCTGAGGGGGAGAGGGACG TGAGAGAGCATGCCTTCTTCCGGAGGATCGACTGGGAAAAACTGGAGAACAGGGAGATCCAGCCACCATTCAAGCCC AAAGTGTGTGGCAAAGGAGCAGAGAACTTTGACAAGTTCTTCACACGAGGACAGCCCGTCTTAACACCACCTGATCA GCTGGTTATTGCTAACATAGACCAGTCTGATTTTGAAGGGTTCTCGTATGTCAACCCCCAGTTTGTGCACCCCATCT TACAGAGTGCAGTAGTT
SEQ ID NO:450 (human kinase protein C α (3KCD) (aminoacid sequence)
MADVFPGNDSTASQDVANRFARKGALRQKNVHEVKDHKFIARFFKQPTFCSHCTDFIWGFGKQGFQCQVCCFVVHKR CHEFVTFSCPGADKGPDTDDPRSKHKFKIHTYGSPTFCDHCGSLLYGLIHQGMKCDTCDMNVHKQCVINVPSLCGMD HTEKRGRIYLKAEVADEKLHVTVRDAKNLIPMDPNGLSDPYVKLKLIPDPKNESKQKTKTIRSTLNPQWNESFTFKL KPSDKDRRLSVEIWDWDRTTRNDFMGSLSFGVSELMKMPASGWYKLLNQEEGEYYNVPIPEGDEEGNMELRQKFEKA KLGPAGNKVISPSEDRKQPSNNLDRVKLTDFNFLMVLGKGSFGKVMLADRKGTEELYAIKILKKDVVIQDDDVECTM VEKRVLALLDKPPFLTQLHSCFQTVDRLYFVMEYVNGGDLMYHIQQVGKFKEPQAVFYAAEISIGLFFLHKRGIIYR DLKLDNVMLDSEGHIKIADFGMCKEHMMDGVTTRTFCGTPDYIAPEIIAYQPYGKSVDWWAYGVLLYEMLAGQPPFD GEDEDELFQSIMEHNVSYPKSLSKEAVSICKGLMTKHPAKRLGCGPEGERDVREHAFFRRIDWEKLENREIQPPFKP KVCGKGAENFDKFFTRGQPVLTPPDQLVIANIDQSDFEGFSYVNPQFVHPILQSAVV
SEQ ID NO:451 (human glucocorticoid receptor (GR) (nucleotide sequence)
ATGGACTCCAAAGAATCATTAACTCCTGGTAGAGAAGAAAACCCCAGCAGTGTGCTTGCTCAGGAGAGGGGAGATGT GATGGACTTCTATAAAACCCTAAGAGGAGGAGCTACTGTGAAGGTTTCTGCGTCTTCACCCTCACTGGCTGTCGCTT CTCAATCAGACTCCAAGCAGCGAAGACTTTTGGTTGATTTTCCAAAAGGCTCAGTAAGCAATGCGCAGCAGCCAGAT CTGTCCAAAGCAGTTTCACTCTCAATGGGACTGTATATGGGAGAGACAGAAACAAAAGTGATGGGAAATGACCTGGG ATTCCCACAGCAGGGCCAAATCAGCCTTTCCTCGGGGGAAACAGACTTAAAGCTTTTGGAAGAAAGCATTGCAAACC TCAATAGGTCGACCAGTGTTCCAGAGAACCCCAAGAGTTCAGCATCCACTGCTGTGTCTGCTGCCCCCACAGAGAAG GAGTTTCCAAAAACTCACTCTGATGTATCTTCAGAACAGCAACATTTGAAGGGCCAGACTGGCACCAACGGTGGCAA TGTGAAATTGTATACCACAGACCAAAGCACCTTTGACATTTTGCAGGATTTGGAGTTTTCTTCTGGGTCCCCAGGTA AAGAGACGAATGAGAGTCCTTGGAGATCAGACCTGTTGATAGATGAAAACTGTTTGCTTTCTCCTCTGGCGGGAGAA GACGATTCATTCCTTTTGGAAGGAAACTCGAATGAGGACTGCAAGCCTCTCATTTTACCGGACACTAAACCCAAAAT TAAGGATAATGGAGATCTGGTTTTGTCAAGCCCCAGTAATGTAACACTGCCCCAAGTGAAAACAGAAAAAGAAGATT TCATCGAACTCTGCACCCCTGGGGTAATTAAGCAAGAGAAACTGGGCACAGTTTACTGTCAGGCAAGCTTTCCTGGA GCAAATATAATTGGTAATAAAATGTCTGCCATTTCTGTTCATGGTGTGAGTACCTCTGGAGGACAGATGTACCACTA TGACATGAATACAGCATCCCTTTCTCAACAGCAGGATCAGAAGCCTATTTTTAATGTCATTCCACCAATTCCCGTTG GTTCCGAAAATTGGAATAGGTGCCAAGGATCTGGAGATGACAACTTGACTTCTCTGGGGACTCTGAACTTCCCTGGT CGAACAGTTTTTTCTAATGGCTATTCAAGCCCCAGCATGAGACCAGATGTAAGCTCTCCTCCATCCAGCTCCTCAAC AGCAACAACAGGACCACCTCCCAAACTCTGCCTGGTGTGCTCTGATGAAGCTTCAGGATGTCATTATGGAGTCTTAA CTTGTGGAAGCTGTAAAGTTTTCTTCAAAAGAGCAGTGGAAGGACAGCACAATTACCTATGTGCTGGAAGGAATGAT TGCATCATCGATAAAATTCGAAGAAAAAACTGCCCAGCATGCCGCTATCGAAAATGTCTTCAGGCTGGAATGAACCT GGAAGCTCGAAAAACAAAGAAAAAAATAAAAGGAATTCAGCAGGCCACTACAGGAGTCTCACAAGAAACCTCTGAAA ATCCTGGTAACAAAACAATAGTTCCTGCAACGTTACCACAACTCACCCCTACCCTGGTGTCACTGTTGGAGGTTATT GAACCTGAAGTGTTATATGCAGGATATGATAGCTCTGTTCCAGACTCAACTTGGAGGATCATGACTACGCTCAACAT GTTAGGAGGGCGGCAAGTGATTGCAGCAGTGAAATGGGCAAAGGCAATACCAGGTTTCAGGAACTTACACCTGGATG ACCAAATGACCCTACTGCAGTACTCCTGGATGTTTCTTATGGCATTTGCTCTGGGGTGGAGATCATATAGACAATCA AGTGCAAACCTGCTGTGTTTTGCTCCTGATCTGATTATTAATGAGCAGAGAATGACTCTACCCTGCATGTACGACCA ATGTAAACACATGCTGTATGTTTCCTCTGAGTTACACAGGCTTCAGGTATCTTATGAAGAGTATCTCTGTATGAAAA CCTTACTGCTTCTCTCTTCAGTTCCTAAGGACGGTCTGAAGAGCCAAGAGCTATTTGATGAAATTAGAATGACCTAC ATCAAAGAGCTAGGAAAAGCCATTGTCAAGAGGGAAGGAAACTCCAGCCAGAACTGGCAGCGGTTTTATCAACTGAC AAAACTCTTGGATTCTATGCATGAAGTGGTTGAAAATCTCCTTAACTATTGCTTCCAAACATTTTTGGATAAGACCA TGAGTATTGAATTCCCCGAGATGTTAGCTGAAATCATCACCAATCAGATACCAAAATATTCAAATGGAAATATCAAA AAACTTCTGTTTCATCAAAAGGTT
SEQ ID NO:452 (human glucocorticoid receptor (GR) (aminoacid sequence)
MDSKESLTPGREENPSSVLAQERGDVMDFYKTLRGGATVKVSASSPSLAVASQSDSKQRRLLVDFPKGSVSNAQQPD LSKAVSLSMGLYMGETETKVMGNDLGFPQQGQISLSSGETDLKLLEESIANLNRSTSVPENPKSSASTAVSAAPTEK EFPKTHSDVSSEQQHLKGQTGTNGGNVKLYTTDQSTFDILQDLEFSSGSPGKETNESPWRSDLLIDENCLLSPLAGE DDSFLLEGNSNEDCKPLILPDTKPKIKDNGDLVLSSPSNVTLPQVKTEKEDFIELCTPGVIKQEKLGTVYCQASFPG ANIIGNKMSAISVHGVSTSGGQMYHYDMNTASLSQQQDQKPIFNVIPPIPVGSENWNRCQGSGDDNLTSLGTLNFPG RTVFSNGYSSPSMRPDVSSPPSSSSTATTGPPPKLCLVCSDEASGCHYGVLTCGSCKVFFKRAVEGQHNYLCAGRND CIIDKIRRKNCPACRYRKCLQAGMNLEARKTKKKIKGIQQATTGVSQETSENPGNKTIVPATLPQLTPTLVSLLEVI EPEVLYAGYDSSVPDSTWRIMTTLNMLGGRQVIAAVKWAKAIPGFRNLHLDDQMTLLQYSWMFLMAFALGWRSYRQS SANLLCFAPDLIINEQRMTLPCMYDQCKHMLYVSSELHRLQVSYEEYLCMKTLLLLSSVPKDGLKSQELFDEIRMTY IKELGKAIVKREGNSSQNWQRFYQLTKLLDSMHEVVENLLNYCFQTFLDKTMSIEFPEMLAEIITNQIPKYSNGNIK KLLFHQKV
SEQ ID NO:453 (L27V02-GR/L27V02-PKCa joint (nucleotide sequence)
GGTGGTTCAGGTGGTGGCGGGAGCGGTGGAGGGAGCAGCGGTGGAGCGATCGCC
SEQ ID NO:454 (L27V02-GR/L27V02-PKCa joint (aminoacid sequence)
GGSGGGGSGGGSSGGAIA
SEQ ID NO:455 (GR-L27V02/PKCa-L27V02 joint (nucleotide sequence)
TCTCTCGGTGGTTCAGGTGGTGGCGGGAGCGGTGGAGGGAGCAGCGGTGGA
SEQ ID NO:456 (GR-L27V02/PKCa-L27V02 joint (aminoacid sequence)
SLGGSGGGGSGGGSSGG
SEQ ID NO:457 (GSSG joint (nucleotide sequence)
GCTCGAGCGGA
SEQ ID NO:458 (GSSG joint (aminoacid sequence)
GSSG
SEQ ID NO:459 (AT1R (nucleotide sequence)
ATGATTCTCAACTCTTCTACTGAAGATGGTATTAAAAGAATCCAAGATGATTGTCCCAAAGCTGGAAGGCATAATTA CATATTTGTCATGATTCCTACTTTATACAGTATCATCTTTGTGGTGGGAATATTTGGAAACAGCTTGGTGGTGATAG TCATTTACTTTTATATGAAGCTGAAGACTGTGGCCAGTGTTTTTCTTTTGAATTTAGCACTGGCTGACTTATGCTTT TTACTGACTTTGCCACTATGGGCTGTCTACACAGCTATGGAATACCGCTGGCCCTTTGGCAATTACCTATGTAAGAT TGCTTCAGCCAGCGTCAGTTTCAACCTGTACGCTAGTGTGTTTCTACTCACGTGTCTCAGCATTGATCGATACCTGG CTATTGTTCACCCAATGAAGTCCCGCCTTCGACGCACAATGCTTGTAGCCAAAGTCACCTGCATCATCATTTGGCTG CTGGCAGGCTTGGCCAGTTTGCCAGCTATAATCCATCGAAATGTATTTTTCATTGAGAACACCAATATTACAGTTTG TGCTTTCCATTATGAGTCCCAAAATTCAACCCTTCCGATAGGGCTGGGCCTGACCAAAAATATACTGGGTTTCCTGT TTCCTTTTCTGATCATTCTTACAAGTTATACTCTTATTTGGAAGGCCCTAAAGAAGGCTTATGAAATTCAGAAGAAC AAACCAAGAAATGATGATATTTTTAAGATAATTATGGCAATTGTGCTTTTCTTTTTCTTTTCCTGGATTCCCCACCA AATATTCACTTTTCTGGATGTATTGATTCAACTAGGCATCATACGTGACTGTAGAATTGCAGATATTGTGGACACGG CCATGCCTATCACCATTTGTATAGCTTATTTTAACAATTGCCTGAATCCTCTTTTTTATGGCTTTCTGGGGAAAAAA TTTAAAAGATATTTTCTCCAGCTTCTAAAATATATTCCCCCAAAAGCCAAATCCCACTCAAACCTTTCAACAAAAAT GAGCACGCTTTCCTACCGCCCCTCAGATAATGTAAGCTCATCCACCAAGAAGCCTGCACCATGTTTTGAGGTTGAGT GA
SEQ ID NO:460 (AT1R (aminoacid sequence)
MILNSSTEDGIKRIQDDCPKAGRHNYIFVMIPTLYSIIFVVGIFGNSLVVIVIYFYMKLKTVASVFLLNLALADLCF LLTLPLWAVYTAMEYRWPFGNYLCKIASASVSFNLYASVFLLTCLSIDRYLAIVHPMKSRLRRTMLVAKVTCIIIWL LAGLASLPAIIHRNVFFIENTNITVCAFHYESQNSTLPIGLGLTKNILGFLFPFLIILTSYTLIWKALKKAYEIQKN KPRNDDIFKIIMAIVLFFFFSWIPHQIFTFLDVLIQLGIIRDCRIADIVDTAMPITICIAYFNNCLNPLFYGFLGKK FKRYFLQLLKYIPPKAKSHSNLSTKMSTLSYRPSDNVSSSTKKPAPCFEVE
SEQ ID NO:461 (IL6-00 signal peptide (nucleotide sequence)
ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTT CCCTGCCCCA
SEQ ID NO:462 (IL6-00 signal peptide (aminoacid sequence)
MNSFSTSAFGPVAFSLGLLLVLPAAFPAP
SEQ ID NO:463 (IL6-00L27V-00 (nucleotide sequence)
ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTT CCCTGCCCCAGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTCC TTGAGCAGGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGC GGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGATCAGATGGGCCAGAT CGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATTCTGCACTATGGCACACTGGTAA TCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAG ATCACTGTAACCGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCT GTTCCGCGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCG
SEQ ID NO:464 (IL6-00L27V-00 (aminoacid sequence)
MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLS GENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKK ITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:465 (native secretion signal+L27V (nucleotide sequence)
atggcttactccacactgttcatcattgctctcacagccgtcgtaacacaagcctcctccacacagaaaagcaacct gacaGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAGC AGGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAA AACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGATCAGATGGGCCAGATCGAAAA AATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATTCTGCACTATGGCACACTGGTAATCGACG GGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACT GTAACCGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCG CGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCG
SEQ ID NO:466 (native secretion signal+L27V (aminoacid sequence)
MAYSTLFIIALTAVVTQASSTQKSNLTVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGE NGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKIT VTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:467 (CP6L27V (RIIbB) (nucleotide sequence) (nucleotide sequence)
Atgttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagttt gtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgaca tccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtac cctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgat cgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtgga acggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtg accggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTT TATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAG TATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTG AAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGG ACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAAT GTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACC TATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTC Tatggtcttcacactcgaagat
SEQ ID NO:468 (CP6L27V (RIIbB) (aminoacid sequence)
MFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVY PVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV TGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEV KITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIAT YEEQLVALYGTNMDIVGSGGSGGSSSMVFTLED
SEQ ID NO:469 (CP12L27V (RIIbB) (nucleotide sequence) (nucleotide sequence)
ATGcagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggt gtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgt atgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggcc gtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcg acgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGC GAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATT CCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAAC AAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGA AAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCT TGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGC AAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTT GCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcga agatttcgttggggactggcga
SEQ ID NO:470 (CP12L27V (RIIbB) (aminoacid sequence)
MQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLC ERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKR KGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLV ALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWR
SEQ ID NO:471 (CP48L27V (RIIbB) (nucleotide sequence)
ATGggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggcca gatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactgg taatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa aagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccct gctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTT CAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTG AAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTC TTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTG CAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCT TCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCAT GGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAG GGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagcc ggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactcc gatccaaaggattgtcctgagc
SEQ ID NO:472 (CP48L27V (RIIbB) (aminoacid sequence)
MGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGK KITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARL KVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAA SAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTA GYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLS
SEQ ID NO:473 (CP52L27V (RIIbB) (nucleotide sequence)
ATGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAAT TTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGG TTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTA ACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGT AACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCTAGCTCAAGCGGAGGTTCAGGCGGTTCCG GAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGAT GTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTAT TGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCG CTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCC ATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAA AAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGAT CTGGAGGTAGCTCTTCTATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTG GACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGAT TGTCCTGAGCGGTGAAAATGGG
SEQ ID NO:474 (CP52L27V (RIIbB) (aminoacid sequence)
MLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITV TGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVD VIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHA IGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENG
SEQ ID NO:475 (CP55L27V (RIIbB) (nucleotide sequence)
Atggacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggt ggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccga acatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggacc ctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaa cggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATG AAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGC ACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGG AGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCT CGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACT GTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACAT CGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTA GCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgag cggtgaaaatgggctgaagatc
SEQ ID NO:476 (CP55L27V (RIIbB) (aminoacid sequence)
MDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGT LWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIG TKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGT VKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQV LEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKI
SEQ ID NO:477 (CP83L27V (RIIbB) (nucleotide sequence)
ATGgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgat cgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtgga acggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtg accggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTT TATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAG TATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTG AAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGG ACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAAT GTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACC TATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTC Tatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaac agggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaa aatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaa aatttttaaggtggtgtaccct
SEQ ID NO:478 (CP83L27V (RIIbB) (aminoacid sequence)
MVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV TGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEV KITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIAT YEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGE NGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYP
SEQ ID NO:479 (CP84L27V (RIIbB) (nucleotide sequence)
atggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcga ctatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacg gcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgacc ggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTAT TGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTAT ACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAA ATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACA GTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTT TAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTAT GAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTat ggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagg gaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaat gggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaat ttttaaggtggtgtaccctgtg
SEQ ID NO:480 (CP84L27V (RIIbB) (aminoacid sequence)
MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT GWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVK ITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATY EEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGEN GLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV
SEQ ID NO:481 (CP103L27V (RIIbB) (nucleotide sequence)
ATGacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgt aacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgag taaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCC GGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGA TGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTA TTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATC GCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGC CATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGA AAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGA TCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacct ggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaagga ttgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaa atgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatgg cacactggtaatcgacggggtt
SEQ ID NO:482 (CP103L27V (RIIbB) (aminoacid sequence)
MTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGS GMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEI ARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGG SGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQ MGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
SEQ ID NO:483 (CP120L27V (RIIbB) (nucleotide sequence)
ATGttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaa ccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtA GCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAG TTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGG AGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAG TGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAAC AAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCT TCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGA ACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggac tggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcgg ggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcc cgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcat cactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacg gccgtatgaaggcatcgccgtg
SEQ ID NO:484 (CP120L27V (RIIbB) (aminoacid sequence)
MFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLE FSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTN KPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGD WRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDH HFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAV
SEQ ID NO:485 (CP123L27V (RIIbB) (nucleotide sequence)
ATGaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacgg ctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCG GAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCA CGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGC TGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGA ATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGA GCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACC TTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATA TTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacag acagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgt aactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaag gtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatga aggcatcgccgtgttcgacggc
SEQ ID NO:486 (CP123L27V (RIIbB) (aminoacid sequence)
MKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSA RLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPR AASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQ TAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDG
SEQ ID NO:487 (CP124L27V (RIIbB) (nucleotide sequence)
ATGaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctc cctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAG GTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGC CTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGA TTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATG GTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCA GCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTG CATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTG TAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagaca gccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaac tccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtc tgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaagg catcgccgtgttcgacggcaaa
SEQ ID NO:488 (CP124L27V (RIIbB) (aminoacid sequence)
MKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSAR LKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRA ASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQT AGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKV ILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGK
SEQ ID NO:489 (CP125L27V (RIIbB) (nucleotide sequence)
ATGatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccct gctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTT CAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTG AAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTC TTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTG CAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCT TCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCAT GGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAG GGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagcc ggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactcc gatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctga gcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatc ctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcat cgccgtgttcgacggcaaaaag
SEQ ID NO:490 (CP125L27V (RIIbB) (aminoacid sequence)
MITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARL KVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAA SAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTA GYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVI LHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKK
SEQ ID NO:491 (CP130L27V (RIIbB) (nucleotide sequence)
ATGaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaac catcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAA TGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTG ATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGA ATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTC GATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATT GGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAG GAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTG GAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggac caagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgt cctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgg gccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcaca ctggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacgg caaaaagatcactgtaacaggg
SEQ ID NO:492 (CP130L27V (RIIbB) (aminoacid sequence)
MTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDV IGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAI GTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLD QVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGT LVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTG
SEQ ID NO:493 (CP145L27V (RIIbB) (nucleotide sequence)
ATGcccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGC tAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGG AGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAG GGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGA AGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTA ACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGG CTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAAC GAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttgggg actggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcat cccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatc atcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaat tatcgacgagcgcctgatcaac
SEQ ID NO:494 (CP145L27V (RIIbB) (aminoacid sequence)
MPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQ GDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFER LLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLIN
SEQ ID NO:495 (CP148L27V (RIIbB) (nucleotide sequence)
ATGtccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAG CGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTG CACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCG GCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGA GAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTC GAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGA CCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGA TATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgac agacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtcc gtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatga aggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttta aggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtat gaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacga gcgcctgatcaaccccgacggc
SEQ ID NO:496 (CP148L27V (RIIbB) (aminoacid sequence)
MSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDS ADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLG PCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVS VTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPY EGIAVFDGKKITVTGTLWNGNKIIDERLINPDG
SEQ ID NO:497 (CP157L27V (RIIbB) (nucleotide sequence)
AtGGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCTAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTA TGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAG GCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCT GGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATG CTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGA CTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAAC ATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGG TAGCTCTTCTATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAG TCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTG AGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCA GATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACACTGG TAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAA AAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCT GCTGTTCCGAGTAACCATCAAC
SEQ ID NO:498 (CP157L27V (RIIbB) (aminoacid sequence)
MGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIES GEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRN IATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVL SGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGK KITVTGTLWNGNKIIDERLINPDGSLLFRVTIN
SEQ ID NO:499 (SS6L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcact gccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatg gagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatg aaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttgg agagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatgg atgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacag ttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTttcgttgggga ctggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcg gggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatca tcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:500 (SS6L27V (RIIbB) (aminoacid sequence)
MVFTLEDSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITM KRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQ LVALYGTNMDIVGSGGSGGSSSFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVII PYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:501 (SS12L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgaAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatga aagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggca ccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatctgga gaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctc gcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactg tcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatc gctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAG CTCttcTcagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcg gggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatca tcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:502 (SS12L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESG EVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNI ATYEEQLVALYGTNMDIVGSGGSGGSSSQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVII PYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:503 (SS26L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctt tggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgct cagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatc agaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaa ctaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaa aggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATgg aacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTggtgtgtccagtttgtttcagaatctcg gggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatca tcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:504 (SS26L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIA QGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFE RLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVII PYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:505 (SS45L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattAGCgctAGCGGAGGTT CAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctg aaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattc ttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtg cagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagct tctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcat ggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgtag gGTCAGGTGGATCTGGAGGtAGCTCttcTgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatca tcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:506 (SS45L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRISASGGSGGSGMYESFIESLPFLKSLEFSARL KVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAA SAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSVLSGENGLKIDIHVII PYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:507 (SS48L27V (RIIbB) (nucleotide sequence)
atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcAGCgctA GCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttct gcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattc ggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaag agaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacct cgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctggg accttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatgg atattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTggtgaaaatgggctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatca tcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:508 (SS48L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSSASGGSGGSGMYESFIESLPFLKSLEFS ARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKP RAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSGENGLKIDIHVII PYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:509 (SS52L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatct ttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgc tcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaat cagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggta actaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttga aaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATg gaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatca tcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:510 (SS52L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGSASGGSGGSGMYESFIESLPFLKS LEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALV TNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSLKIDIHVII PYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:511 (SS55L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattc cttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaaca aatcattgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaa agggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagctt gccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgca agcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttg ccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTgacatccatgtcatcatc ccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatca tcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:512 (SS55L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKISASGGSGGSGMYESFIESLPF LKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGEL ALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSDIHVII PYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:513 (SS83L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcact gccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatg gagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatg aaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttgg agagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatgg atgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacag ttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTgtggatgatca tcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:514 (SS83L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITM KRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQ LVALYGTNMDIVGSGGSGGSSSVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:515 (SS84L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtgAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtc actgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacg atggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattact atgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactt tggagagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaa tggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaa cagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTgatgatca tcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:516 (SS84L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKIT MKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEE QLVALYGTNMDIVGSGGSGGSSSDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:517 (SS85L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattga gtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtataca acgatggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaatt actatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagta ctttggagagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttag caatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaa gaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTgatca tcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:518 (SS85L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKI TMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYE EQLVALYGTNMDIVGSGGSGGSSSDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:519 (SS100L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcAGCgc tAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagtttt ctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagat tcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtgga agagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaac ctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctg ggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacat ggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTgacggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:520 (SS100L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVISASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGD SADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLL GPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:521 (SS101L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacAG CgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagt tttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcaggga gattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagt ggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaaca aacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggctt ctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaa catggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTggggttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:522 (SS101L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQG DSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERL LGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:523 (SS102L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg gAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttgg agttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcag ggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcaga agtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaacta acaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaagg cttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaac gaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTgttacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:524 (SS102L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQ GDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFER LLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:525 (SS103L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctt tggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgct cagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatc agaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaa ctaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaa aggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATgg aacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTacgccgaacatgatcgactatttcggac ggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:526 (SS103L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIA QGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFE RLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:527 (SS120L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgAGCgctAGCGGAGGTTCAGGCG GTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagta gtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattctttttt cattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtag aaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagcttctgcc cacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaat tatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAG GTGGATCTGGAGGtAGCTCttcTttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:528 (SS120L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVSASGGSGGSGMYESFIESLPFLKSLEFSARLKV VDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASA HAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:529 (SS123L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcAGCgctAGCGGAG GTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgc ctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctga ttcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatg gtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagca gcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttg catggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggatattg taggGTCAGGTGGATCTGGAGGtAGCTCttcTaaaaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:530 (SS123L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGSASGGSGGSGMYESFIESLPFLKSLEFSAR LKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRA ASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:531 (SS124L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaAGCgctAGCG GAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgca cgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggc tgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagaga atggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcga gcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggacc ttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggata ttgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTaagatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:532 (SS124L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKSASGGSGGSGMYESFIESLPFLKSLEFSA RLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPR AASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:533 (SS125L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagAGCgctA GCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttct gcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattc ggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaag agaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacct cgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctggg accttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatgg atattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTatcactgtaacagggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:534 (SS125L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKSASGGSGGSGMYESFIESLPFLKSLEFS ARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKP RAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:535 (SS130L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taacagggAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaa tctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcat tgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggta aatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctg gtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatt tgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgT ATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:536 (SS130L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGSASGGSGGSGMYESFIESLPFLK SLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELAL VTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:537 (SS136L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taacagggaccctgtggaacggcaacAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgag tcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaa cgatggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaatta ctatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtac tttggagagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagc aatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaag aacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:538 (SS136L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNSASGGSGGSGMYESFIE SLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQY FGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSKI IDERLINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:539 (SS143L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgAGCgctAGCGGAGGTTCAGGCGGTTCCGGA atgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgt gataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttcattattg aatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgct cgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccat tgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaa ggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCT GGAGGtAGCTCttcTatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:540 (SS143L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLSASGGSGGSG MYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIA RCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGS GGSSSINPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:541 (SS145L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacAGCgctAGCGGAGGTTCAGGCGGT TCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagt agatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttca ttattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaa atcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagcttctgccca cgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaatta tgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGT GGATCTGGAGGtAGCTCttcTcccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:542 (SS145L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINSASGGSGG SGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVE IARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSG GSGGSSSPDGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:543 (SS147L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacAGCgctAGCGGAGGTTCA GGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaa agtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattctt ttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgca gtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagcttc tgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatgg aaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgtaggG TCAGGTGGATCTGGAGGtAGCTCttcTggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:544 (SS147L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDSASGGS GGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGA VEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVG SGGSGGSSSGSLLFRVTINGVTGWRLCERILA
SEQ ID NO:545 (SS148L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggcAGCgctAGCGGAGGT TCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcct gaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgatt cttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggt gcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagc ttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgca tggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgta ggGTCAGGTGGATCTGGAGGtAGCTCttcTtccctgctgttccgagtaaccatcaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:546 (SS148L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSASGG SGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENG AVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIV GSGGSGGSSSSLLFRVTINGVTGWRLCERILA
SEQ ID NO:547 (SS156L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccga gtaaccatcAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaa atctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatca ttgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggt aaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccct ggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcat ttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctg TATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTaacggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:548 (SS156L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTISASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKG KSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVAL YGTNMDIVGSGGSGGSSSNGVTGWRLCERILA
SEQ ID NO:549 (SS157L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccga gtaaccatcaacAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattcct taaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaa tcattgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaag ggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgc cctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaag catttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgcc ctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTggagtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:550 (SS157L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRK GKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVA LYGTNMDIVGSGGSGGSSSGVTGWRLCERILA
SEQ ID NO:551 (SS158L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccga gtaaccatcaacggaAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccatt ccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaac aaatcattgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaaga aagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagct tgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgc aagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagtt gccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTgtgaccggctggcggct gtGCGAACGCATTCTGGCt
SEQ ID NO:552 (SS158L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKR KGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLV ALYGTNMDIVGSGGSGGSSSVTGWRLCERILA
SEQ ID NO:553 (SS166L27V (RIIbB) (nucleotide sequence)
ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaaca gggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaa atgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgg ggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactg taacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccga gtaaccatcaacggagtgaccggctggcggctgtGCGAAAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatga aagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggca ccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatctgga gaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctc gcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactg tcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatc gctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAG CTCttcTCGCATTCTGGCt
SEQ ID NO:554 (SS166L27V (RIIbB) (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCESASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESG EVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNI ATYEEQLVALYGTNMDIVGSGGSGGSSSRILA
SEQ ID NO:555 (CP26L27V (RIIbB) (nucleotide sequence)
ATGggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaa tgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaa tttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggg gttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgt aacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgag taaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCC GGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGA TGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTA TTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATC GCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGC CATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGA AAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGA TCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacct ggaccaagtccttgaacaggga
SEQ ID NO:556 (CP26L27V (RIIbB) (aminoacid sequence)
MGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDG VTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGS GMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEI ARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGG SGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQG
SEQ ID NO:557 (CP45L27V (RIIbB) (nucleotide sequence)
ATGgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgacca aatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatg gcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttc gacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccga cggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAA GCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCT GCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTC GGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAG AGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCT CGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGG ACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGG ATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcga cagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtc cgtaactccgatccaaaggatt
SEQ ID NO:558 (CP45L27V (RIIbB) (aminoacid sequence)
MVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVF DGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFS ARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKP RAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWR QTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI
SEQ ID NO:559 (CP100 (+1) L27V (RIIbB) (nucleotide sequence)
ATGgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaa gatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgc tgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCA GGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAA AGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTT TTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCA GTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTC TGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGG AAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGG TCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccgg ctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccga tccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagc ggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcct gcactatggcacactggtaatcGAC
SEQ ID NO:560 (CP100 (+1) L27V (RIIbB) (aminoacid sequence)
MDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGS GGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGA VEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVG SGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLS GDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVID
SEQ ID NO:561 (CP101 (+1) L27V (RIIbB) (nucleotide sequence)
ATGggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagat cactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgt tccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGC GGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGT AGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTT TCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTA GAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGC CCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAA TTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCA GGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccggcta caacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatcc aaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgca ctatggcacactggtaatcgacGGG
SEQ ID NO:562 (CP101 (+1) L27V (RIIbB) (aminoacid sequence)
MGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSG GSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAV EIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGS GGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDG
SEQ ID NO:563 (CP102 (+1) L27V (RIIbB) (nucleotide sequence)
ATGgttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcac tgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttcc gagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGT TCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGT AGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCA TTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAA ATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCA CGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTA TGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGT GGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaa cctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaa ggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgac caaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcacta tggcacactggtaatcgacgggGTT
SEQ ID NO:564 (CP102 (+1) L27V (RIIbB) (aminoacid sequence)
MVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGG SGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVE IARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSG GSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGD QMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
SEQ ID NO:565 (CP143 (+1) L27V (RIIbB) (nucleotide sequence)
ATGatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCAT TCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAAT CTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATT GCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAA ATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGG TAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTT GAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTA TGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcg ttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcag aatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgt catcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtgg atgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactat ttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaa caaaattatcgacgagcgcctgATC
SEQ ID NO:566 (CP143 (+1) L27V (RIIbB) (aminoacid sequence)
MINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQII AQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAF ERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQ NLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDY FGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLI
SEQ ID NO:567 (CP147 (+1) L27V (RIIbB) (nucleotide sequence)
ATGggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTC AAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTT CTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGAT TCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGA AGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAAC CTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTG GGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACAT GGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggc gacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgta tgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcact ttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcga cgagcgcctgatcaaccccgacGGC
SEQ ID NO:568 (CP147 (+1) L27V (RIIbB) (aminoacid sequence)
MGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGD SADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLL GPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGV SVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRP YEGIAVFDGKKITVTGTLWNGNKIIDERLINPDG
SEQ ID NO:569 (CP156 (+1) L27V (RIIbB) (nucleotide sequence)
ATGaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAAT GTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGA TAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAA TCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCG ATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTG GGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGG AACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGG AGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggacc aagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtc ctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatggg ccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacac tggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc aaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctc cctgctgttccgagtaaccatcAAC
SEQ ID NO:570 (CP156 (+1) L27V (RIIbB) (aminoacid sequence)
MNGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIE SGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKR NIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIV LSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDG KKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN
SEQ ID NO:571 (CP158 (+1) L27V (RIIbB) (nucleotide sequence)
ATGgtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGA AAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCA CCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGA GAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTC GCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTG TCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATC GCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAG CTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtcc ttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagc ggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagat cgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaa tcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag atcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgct gttccgagtaaccatcaacggaGTG
SEQ ID NO:572 (CP158 (+1) L27V (RIIbB) (aminoacid sequence)
MVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESG EVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNI ATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLS GENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKK ITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV
SEQ ID NO:573 (CP166 (+1) L27V (RIIbB) (nucleotide sequence)
ATGCGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATT CCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAAC AAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGA AAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCT TGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGC AAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTT GCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcga agatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtt tgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgac atccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgta ccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatga tcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtgg aacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagt gaccggctggcggctgtGCGAACGC
SEQ ID NO:574 (CP166 (+1) L27V (RIIbB) (aminoacid sequence)
MRILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKR KGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLV ALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKID IHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLW NGNKIIDERLINPDGSLLFRVTINGVTGWRLCER
SEQ IDNO:575 (joint)
GGGTCAGGTGGATCTGGAGGTAGCTCTTCT
SEQ ID NO:576 (joint)
AGCTCAAGCGGAGGTTCAGGCGGTTCCGGA
SEQ ID NO:577 (LC3 (nucleotide sequence))
ATGCCGTCCGAGAAGACCTTCAAACAGCGCCGGAGCTTCGAACAAAGAGTGGAAGATGTCCGGCTCATCCGGGAGCA GCACCCCACCAAGATCCCAGTGATTATAGAGCGATACAAGGGTGAGAAGCAGCTGCCCGTCCTGGACAAGACCAGTT CCTTGTACCTGATCACGTGAATATGAGCGAACTCATCAAGATAATTAGAAGGCGCCTGCAGCTCAATGCTAACCAAG CCTTCTTCCTCCTGGTGAATGGGCACAGCATGGTGAGTGTGTCCACACCCATCTCTGAAGTGTACGAGAGCGAGAGA GATGAAGACGGCTTCCTGTACATGGTCTATGCCTCCCAGGAGACGTTCGGGACAGCACTGGCTGTGTAA
SEQ ID NO:578 (LC3 (aminoacid sequence))
MPSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNMSELIKIIRRRLQLNANQ AFFLLVNGHSMVSVSTPISEVYESERDEDGFLYMVYASQETFGTALAV
SEQ ID NO:579 (mode sequences) GSAIVK
SEQ ID NO:580 (mode sequences) NHGK
SEQ ID NO:581 (mode sequences) SILM
SEQ ID NO:582 (mode sequences) AVTK
SEQ ID NO:583 (mode sequences) LIVMFY
SEQ ID NO:584 (mode sequences) LIVMFY
SEQ ID NO:585 (mode sequences) LIVM
SEQ ID NO:586 (mode sequences) LIVMWF
SEQ ID NO:587 (mode sequences) EKTQ
SEQ ID NO:588 (mode sequences) LIVMAKR
SEQ ID NO:589 (mode sequences)
LIVMAKRG
SEQ ID NO:590 (mode sequences)
LIVMF
SEQ ID NO:591 (mode sequences)
LIVMFSYQ
SEQ ID NO:592OgLuc L27V-LC3 fusant albumen (nucleotide sequence)
ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAGCA GGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAA ACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGATCAGATGGGCCAGATCGAAAAA ATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATTCTGCACTATGGCACACTGGTAATCGACGG GGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTG TAACCGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGC GTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGAGAATTTTGGCGGGCTCGAGCGGAGGTGGAGGTTCAGG TGGTGGCGGGAGCGGTGGAATGCCGTCCGAGAAGACCTTCAAACAGCGCCGGAGCTTCGAACAAAGAGTGGAAGATG TCCGGCTCATCCGGGAGCAGCACCCCACCAAGATCCCAGTGATTATAGAGCGATACAAGGGTGAGAAGCAGCTGCCC GTCCTGGACAAGACCAAGTTCCTTGTACCTGATCACGTGAATATGAGCGAACTCATCAAGATAATTAGAAGGCGCCT GCAGCTCAATGCTAACCAAGCCTTCTTCCTCCTGGTGAATGGGCACAGCATGGTGAGTGTGTCCACACCCATCTCTG AAGTGTACGAGAGCGAGAGAGATGAAGACGGCTTCCTGTACATGGTCTATGCCTCCCAGGAGACGTTCGGGACAGCA CTGGCTGTGTAA
SEQ ID NO:593OgLuc L27V-LC3 fusant albumen (aminoacid sequence)
MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAGSSGGGGSGGGGSGGMPSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLP VLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVSVSTPISEVYESERDEDGFLYMVYASQETFGTA LAV

Claims (26)

1. the compound of a formula (Ia):
Wherein R2Group selected from consisting of:
Or；
R6Choosing freely-H ,-OH ,-NH2,-OC (O) R or-OCH2The group of OC (O) R composition；
R8Choosing is freely, H or C3-5The group of cycloalkyl composition；
X is-S-,-O-or-NR22-；
Z is-CH-or-N-；
R22For H；
Each R independently be C1-7Straight chained alkyl or C3-7Branched alkyl；
The key table of dotted line shows the existence of optional ring, and it can be saturated or unsaturated；
Precondition is to work as R6For NH2Time, R2For；
Or R8It is not。
Compound the most according to claim 1, wherein R2For；And X is O or S.
3. according to compound in any one of the preceding claims wherein, wherein R8For C3-5Cycloalkyl or H.
4. according to the compound according to any one of claim 1-2, wherein R8It is。
5. one kind selected from following compound:
。
6. a formulaOrCompound.
7. the compound of a formula (Ia):
Wherein R2Group selected from consisting of:
Or,
R6Choosing freely-H ,-OH ,-NH2,-OC (O) R or-OCH2The group of OC (O) R composition；
R8Choosing freely-CH2C6H5、-C(R3)(R4) H, H or C3-5The group of cycloalkyl composition；
Wherein R3And R4It is H or is C1-2Alkyl；
X is-NR22-；
Z is-CH-or-N-；
R22For CH3Or CH2CH3；
Each R independently be C1-7Straight chained alkyl or C3-7Branched alkyl；
The key table of dotted line shows the existence of optional ring, and it can be saturated or unsaturated；
Precondition is to work as R6For NH2Time, R2For；
Or R8It is not。
Compound the most according to claim 7, wherein R8It is-C (R3)(R4)H、C3-5Cycloalkyl or H.
Compound the most according to claim 7, wherein R8It is-CH2C6H5。
10. one kind selected from following compound:
、Or。
The compound of 11. 1 kinds of formulas (Ia):
Wherein R2Group selected from consisting of:
Or；
R6Choosing freely-H ,-OH ,-NH2,-OC (O) R or-OCH2The group of OC (O) R composition；
R8Choosing freely-CH2C6H5、-C(R3)(R4) H, H or C3-5The group of cycloalkyl composition；
Wherein R3And R4It is H or is C1-2Alkyl；
X is-S-,-O-or-NR22-；
Z is-CH-or-N-；
R22For H；
Each R independently be C1-7Straight chained alkyl or C3-7Branched alkyl；
The key table of dotted line shows the existence of optional ring, and it can be saturated or unsaturated；
Precondition is to work as R6For NH2Time, R2For；
Or R8It is not-CH2C6H5。
12. compounds according to claim 11,
Wherein R2For；And
X is-O-or-S-.
13. according to the compound according to any one of claim 11-12, wherein R8It is-C (R3)(R4)H、C3-5Cycloalkyl or H.
14. according to the compound according to any one of claim 11-12, wherein R8It is-CH2C6H5。
15. 1 kinds of test kits, including according to the compound according to any one of claim 1-14.
16. test kits according to claim 15, also include luciferase.
17. test kits according to claim 16, wherein said luciferase is that thorn shrimp belongs to or sea pansy belongs to luciferase.
18. according to the test kit according to any one of claim 15-17, also include buffer reagent.
19. 1 kinds are used for detecting the method that sample is luminous, and described method includes:
Sample is contacted with according to the compound according to any one of claim 1-14；
If the described luciferase utilizing coelenterazine is not present in described sample, by described sample and the fluorescence utilizing coelenterazine Element enzyme contacts；With
Detection luciferase.
20. methods according to claim 19, wherein said sample comprises living cells.
21. method according to claim 19, wherein said sample comprises the luciferase utilizing coelenterazine.
22. 1 kinds are used for the method detecting luminescence in transgenic animal, and described method includes
Use according to the compound according to any one of claim 1-14 to transgenic animal；With detection luminescence；
Wherein said transgenic animal express the luciferase utilizing coelenterazine.
23. according to the compound according to any one of claim 1-14 in preparation use in the test kit that diagnoses the illness On the way.
24. purposes according to claim 23, wherein said test kit also includes luciferase.
25. purposes according to claim 24, wherein said luciferase is that thorn shrimp belongs to or sea pansy belongs to luciferase.
26. also include buffer reagent according to the purposes according to any one of claim 23-25, wherein said test kit.
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